key: cord- -tsuz j authors: ngan, luong thi my; jang, myeong jin; kwon, min jung; ahn, young joon title: antiviral activity and possible mechanism of action of constituents identified in paeonia lactiflora root toward human rhinoviruses date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: tsuz j human rhinoviruses (hrvs) are responsible for more than half of all cases of the common cold and cost billions of usd annually in medical visits and missed school and work. an assessment was made of the antiviral activities and mechanisms of action of paeonol (pa) and , , , , -penta-o-galloyl-β-d-glucopyranose (pgg) from paeonia lactiflora root toward hrv- and hrv- in mrc cells using a tetrazolium method and real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. results were compared with those of a reference control ribavirin. based on % inhibitory concentration values, pgg was . and . times more active toward hrv- ( . μm) and hrv- ( . μm) in mrc cells, respectively, than ribavirin. the constituents had relatively high selective index values ( . –> . ). the μg/ml pa and μg/ml pgg did not interact with the hrv- particles. these constituents inhibited hrv- infection only when they were added during the virus inoculation ( h), the adsorption period of hrvs, but not after h or later. moreover, the rna replication levels of hrvs were remarkably reduced in the mrc cultures treated with these constituents. these findings suggest that pgg and pa may block or reduce the entry of the viruses into the cells to protect the cells from the virus destruction and abate virus replication, which may play an important role in interfering with expressions of rhinovirus receptors (intercellular adhesion molecule- and low-density lipoprotein receptor), inflammatory cytokines (interleukin (il)- , il- , tumor necrosis factor, interferon beta, and il- β), and toll-like receptor, which resulted in diminishing symptoms induced by hrv. global efforts to reduce the level of synthetic drugs justify further studies on p. lactiflora root-derived materials as potential anti-hrv products or lead molecules for the prevention or treatment of hrv. human rhinoviruses (hrvs) (picornaviridae) are the most common cause of upper respiratory tract infection (or common cold) and are responsible for more than half of all cases of the common cold [ , ] . they are also associated with more severe diseases such as acute otitis media in children [ ] and sinusitis in adults [ ] . hrvs can also cause severe lower respiratory tract symptoms such as pneumonia, wheezing, bronchiolitis, and exacerbations of asthma and chronic obstructive pulmonary disease in infants and children as well as fatal pneumonia in elderly and immunocompromised adults [ , ] . although hrv-induced upper respiratory illness is often mild and self-limiting, the socioeconomic burden caused by medical visits and missed school and work by hrv infection is enormous [ , , ] . the degree of drug misuse and abuse is significant and antihistamine and antibiotic usages have caused many side effects [ ] . attempts to develop effective treatments or vaccination have been relatively limited and unsuccessful because of more than serotypes of hrv [ , , ] . there is a need for the development of selective antiviral agents with novel target sites to establish an effective hrv management strategy and tactics because currently no effective antiviral therapies have been approved for either the prevention or treatment of diseases caused by hrv infection [ ] . plants may provide potential sources of antiviral products largely because they constitute a potential source of bioactive secondary metabolites that have been perceived by the general public as relatively safe, with minimal impacts to human health, and often act at multiple and novel target sites [ ] [ ] [ ] [ ] . certain plant preparations and their constituents are regarded as potential sources for commercial antiviral products for prevention or treatment of hrv infection. previous studies have shown that a methanol extract from the root of chinese peony, paeonia lactiflora pallas (paeoniaceae), possessed good antiviral activity toward hrv- and hrv- . no work has been obtained concerning the potential use of p. lactiflora to manage hrv, although historically p. lactiflora root ( - g of dried root/ times/day) is used as analgesic, hemostyptic, and bacteriostatic agents [ , ] . the aim of the study was to assess the cytotoxic and antiviral effects on two cell lines (hela and mrc ) and two hrv serotypes (hrv- and hrv- ) of paeonol (pa), gallic acid (ga), and , , , , -penta-o-galloyl-β-d-glucopyranose (pgg) from p. lactiflora root using a -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide (mtt) assay. the antiviral activities of these materials were compared with those of ribavirin, a broad-spectrum antiviral agent currently used clinically to treat various dna and rna virus infections [ ] . the antiviral properties and mechanisms of action of the constituents also were elucidated using real-time quantitative reverse transcription polymerase chain reaction (qrt-pcr) with sybr green dye and specific enzyme-linked immunosorbent assay (elisa). instrumental analysis merck silica gel rp- f s plates (for rp-tlc) and an agilent series high-performance liquid chromatograph (hplc) (santa clara, ca) were used for isolation of active principles. ribavirin (> % purity) and mtt were purchased from tokyo chemical industry (tokyo, japan) and sigma-aldrich (st. louis, mo), respectively. anitbiotic-antimycotic and minimum essential medium (mem) were purchased from invitrogen (grand island, ny). fetal bovine serum (fbs) was supplied by paa laboratories (etobicoke, ontario, canada). the protein molecular weight standards (precision plus protein all blue standards) were purchased from bio-rad life sciences (hercules, ca). ripa buffer and % mammalian cell protease inhibitor cocktail were purchased from sigma-aldrich. the primary antibodies used in this study were as follows: anti-icam- antibody [rabbit polyclonal to intercellular adhesion molecule- (icam- )], anti-low-density lipoprotein receptor (ldlr) antibody (rabbit polyclonal to ldlr), and anti-actin antibody (rabbit polyclonal to actin) purchased from abcam (cambridge, uk). the secondary antibody (horseradish peroxidase conjugated goat polyclonal to rabbit) was supplied by abcam. all of the other chemicals and reagents used in this study were of analytical grade quality and available commercially. hela (atcc ccl- ), a human epithelial adenocarcinoma cervix cell line, and mrc (atcc ccl- ), a human lung fibroblast cell line, were purchased from the american type culture collection (atcc) (manassas, va). these cell lines were maintained in mem supplemented with % fbs and antibiotic-antimycotic solution ( units/l of penicillin, mg/l of streptomycin, and μg/l of amphotericin) in a humidified incubator at °c and % co . hrv- (atcc vr- as/gp) and hrv- (atcc vr- as/gp) were purchased from atcc. virus titers were determined by cytopathic effects (cpe) in hela and mrc cells and were expressed as % tissue culture infective dose (tcid ) per ml as described previously by morgan [ ] . extraction procedures of air-dried root of p. lactiflora were performed as described previously by ngan et al. [ ] . for isolation of active principles, viral cpe inhibition assay [ ] toward hrv- in hela cell was used. the hexane-soluble fraction was most biologically active (table ) and was chromatographed as described previously [ ] . finally, an active principle ( mg) was isolated at a retention time of . min. the other active ethyl acetate-soluble fraction ( g) was chromatographed on a × cm silica gel column ( g) by elution with a gradient of chloroform and methanol [ : ( l) (fig ) by spectroscopic analyses, including ms and nmr. pa ( ): compound was isolated as an antibacterial principle from p. lactiflora root in our previous work [ ] , and the spectral data of compound was largely identical to the published data [ ] . ga ( ) was identified on the basis of the following evidence: white powder. [ ] . pgg ( ): compound was isolated as an antibacterial principle from p. lactiflora root in our previous work [ ] , and the spectral data of compound was largely identical to the published data [ , ] . the cytotoxicity of the test materials to two human cell lines was evaluated using an mtt assay described previously by morgan [ ] . in brief, a × stock solution of mtt was prepared by adding mg/ml mtt in phosphate-buffered saline (pbs) (ph . ). the stock solution was sterile-filtered and stored at − °c. hela and mrc cells were seeded onto -well culture plates at a density of × cells per well for day. the culture medium was removed and the plates with monolayer cells were replaced with media containing several different concentrations ( - μg/ml) of the test materials in dimethylsulfoxide (dmso). after incubation at °c and % co for days, the culture plates were then washed once with μl pbs. a volume of μl medium containing . % mtt were added to each well and then incubated for h at the same condition. after then, mtt solution was removed and μl dmso was added to each well. finally, the plate was shaken for min to dissolve the purple formazan crystals that had formed. absorbance was read at nm by using a versamax microplate reader (molecular devices, sunnyvale, ca) with a reference absorbance at nm. monolayers of hela and mrc cells were seeded onto a -well culture plate as stated previously. subsequently, μl media containing several different concentrations ( - μg/ml) of each test material in dmso was put into the wells, and then μl of ×tcid of the virus stock was added to produce an appropriate cpe within days after infection. ribavirin served as a reference control and was similarly prepared. negative controls consisted of the dmso solution. viral inhibition rate (vir) (%) was calculated according to the formula [ ] , vir = (a tv -a cv )/(a cd -a cv ) × , where a tv is the optical density measured with a given concentration of the test compound in hrv infected cells; a cv is the optical density measured for the control untreated hrv infected cells; a cd is the optical density measured for the control untreated hrv uninfected cells. virus titers in infected mrc- cultures treated with pa and pgg were measured as tcid using mtt-based titration method [ ] . in brief, mrc cells were seeded at cells/ml in a -well plate. after h, the cell monolayers were treated with μg/ml pa or μg/ml pgg and infected with hrv- or hrv- in a concentration of ×tcid /ml. uninfected untreated and infected untreated cultures were included in the assay. the μg/ml of ribavirin was used as a reference control. after h incubation at °c, the cultures were frozen and thawed at − °c/ °c. cell debris was removed by centrifugation ( rpm) and the virus supernatants were collected. virus titration was then performed in hela cell cultures. initially, a : dilution of each supernatant was prepared followed by serial -fold-dilutions, and added to hela cell monolayers. after h, cell mortality was measured using an mtt assay stated previously. graphs were built by plotting dead cell percentages toward virus dilution factors of each virus supernatant. the % infectivity point was calculated through a linear regression analysis of the curve. the effects of the test compounds on the infectivity of hrv- particles were elucidated as described previously by choi et al. [ ] . approximately . -fold quantities of the ic values of each test compound were applied. hrv- was preincubated with μg/ml pa, μg/ml pgg, or μg/ml ribavirin for h at °c. monolayers of mrc cells were infected with the pretreated or untreated hrv- for h at °c. unbound virus was removed by washing the wells with pbs twice, and then cells were incubated in fresh medium supplemented with or without test compounds at °c. after days, mtt test and antiviral activity were carried out as stated previously. the time-of-addition effects of all compounds on hrv- were examined according to the method of choi et al. [ ] . in brief, monolayers of mrc cells were seeded onto a -well culture plate as stated previously. after washing with pbs, μg/ml pa, μg/ml pgg, and μg/ml ribavirin were separately added onto the cells at either before (- h), during ( h), or after ( , , , , , , , , , and h) hrv- infection at °c. after days, mtt test and antiviral activity were carried out as stated previously. to evaluate the level of gene expression, real-time qrt-pcr with sybr green dye was carried out. hrv- or hrv- infected and noninfected cultures of mrc monolayers grown in cm cell culture flasks (corning, ny) were treated with μg/ml pa or μg/ml pgg. after incubation at °c and % co for days, total rna was extracted from the culture cells using the rneasy plus mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. contaminated genomic dna was removed using rq rnase-free dnase (promega, madison, wi). complementary dna (cdna) was synthesized using μg total rna through a reverse transcription reaction using the superscript first-strand synthesis kit (invitrogen, carlsbad, ca). five log -fold dilutions of cdna for each rna were performed to determine pcr efficiency ( ng- pg per reaction). qrt-pcr was performed in -well plates using the steponeplus real-time pcr system (applied biosystems, foster, ca). each reaction mixture consisted of μl of maxima sybr green/rox qpcr master mix ( ×) (thermo scientific, foster, ca), μl of forward and reverse primers ( pmol each), μl of cdna ( ng), and μl of double-distilled water in a final volume of μl. oligonucleotide pcr primer pairs are listed in table cell lysates from infected and noninfected mrc cultures days after incubation with or without test compounds were obtained in ripa buffer and % mammalian cell protease inhibitor cocktail according to the manufacturer's instructions. working dilutions of primary antibodies were dilulted , , and times for anti-icam- , anti-ldlr, and anti-actin antibodies, respectively. working dilutions of secondary antibody was , , and for anti-icam- , anti-ldlr, and anti-actin, respectively. ten micrograms of cell lysates from different treatments were mixed with an equal volume of × laemmli sample buffer, boiled in min, and resolved by electrophoresis in % sodium dodecyl sulfate-polyacrylamide gels (sds-page) [ ] . after electrophoresis at v in h, proteins from the gels were transferred onto a polyvinyl difluoride membrane (pall corporation, pensacola, fl) using a electroblotting apparatus (bio-rad, hercules, ca). the membranes for anti-icam- and anti-ldlr were incubated in blocking solution containing % nonfat dry milk for h to inhibit nonspecific binding. these membranes were then incubated overnight at °c with the primary antibodies. after washing with pbs (each min) three times, the membranes were further incubated with the secondary antibody for h and washed with pbs containing . % tween- (v/v) ( . % pbs-t) four times (each min). the membranes for anti-actin were incubated in pbs blocking solution containing % bsa overnight to inhibit nonspecific binding, and then incubated with anti-actin antibody in the blocking solution for h at °c and washed four times (each min) in . % pbs-t at room temperature before incubation with the second antibody. finally, all the membranes were developed with an ecl chemiluminescence reagent (amersham bioscience, buckinghamshire, uk) and exposed to a cp-pu x-ray film (agfa, mortsel, belgium). differences in protein expressions were quantified using a molecular imager gel doc xr system (bio-rad, hercules, ca) and normalized to actin expression on the same membrane. the expressions of icam- and ldlr were examined using real-time qrt-pcr and western blot. furthermore, concentrations of soluble icam- (sicam- ) in cell-free culture supernatants days after treatment were measured using a sicam- elisa kit (pierce biotechnology, rockford, il) according to the manufacturer's instructions. the sensitivities of the assays were . ng/ml. the concentrations of icam- in the test samples were determined from od values using standard curve of each assay. the concentrations of il- , il- , and tnf in cell-free culture supernatants days after treatment were measured using specific elisa. opteia il- , il- , and tnf elisa kits (bd biosciences, san diego, ca) were used for the assays. the sensitivities of these elisa assays were . , . , and . pg/ml, respectively. the concentrations of il- , il- , and tnf in the test samples were determined from od values using standard curve of each assay. cytotoxicity was expressed as % cytotoxic concentration (cc ) of each compound that reduced the viability of cells to % of the control. fifty percent inhibitory concentration (ic ) was defined as the compound concentration required to reducing the viral cpe to % of the control. the cc and ic values were determined using graphpad prism software program (graphpad software, la jolla, ca). the ic values for each serotype and their treatments were considered to be significantly different from one another when their % confidence limits (cls) did not overlap. the selectivity index (si) was determined as the ratio of cc to ic [ ] . all data represent the mean ± sd of duplicate or triplicate samples of three independent experiments. statistical analyses were carried out using sas . program (sas institute, cary, nc). data from two groups were analyzed by a student's t-test, and multiple groups were analyzed by a one-way analysis of variance and bonferroni multiple comparison post-test. the antiviral activity of pgg, ga, and pa toward hrv- and hrv- in hela cells was compared with that of ribavirin using an mtt assay (table ) . based on ic effects of pa and pgg on hrv titers were compared with those of ribavirin (table ) due to their antiviral activity with high selectivity, the effects of pa and pgg on the infectivity of hrv- particles were likewise compared with those of ribavirin (fig ) . the inhibition rates of preincubation with μg/ml pa, μg/ml pgg, and μg/ml ribavirin were . , . , and . %, respectively. continuous presence of pa, pgg, and ribavirin during infection led to a significant increase in the inhibition rate ( . , . , and . %). to investigate the mode of action of pa, pgg, and ribavirin, time-of-addition experiments were performed (fig ) . treatment with μg/ml pa or μg/ml pgg considerably suppressed hrv- infection only when added just after the virus inoculation ( h) ( . and . % inhibition). the inhibition of these compounds declined to % or less when added at either prior (- h) or post ( - h) infection. similar results were observed with μg/ ml ribavirin. further evidence of the inhibitory effects of pa and pgg on hrv replication in mrc- cells was provided by real-time qrt-pcr analysis (fig ) . in the presence of μg/ml pa or μg/ml pgg in mrc cell cultures infected with hrv- , the rna replication levels were reduced by . and . fold, respectively, compared to the levels in the cell cultures without the compounds (fig a) . similarly, the replication levels of the hrv- in the mrc cell culture treated with pa or pgg were also reduced by . and . fold, respectively, compared with the untreated cultures ( fig b) . unbound viruses were removed and washed by phosphate-buffered saline twice, and then cells were incubated in fresh medium with or without test compounds. after days, inhibition was evaluated by tetrazolium method and expressed as the inhibition rate. each bar represents the mean ± sd of triplicate samples of three independent experiments. ***p< . , using a student's t-test. effect on icam- and ldlr expressions mrna expression of icam- in mrc days after hrv infection in the presence of μg/ml pa or μg/ml pgg were investigated using real-time qrt-pcr (fig a) . hrv- or hrv- infection increased icam- mrna expression, but these increases were reduced by the addition of pa and pgg. furthermore, the icam- mrna expression level in the group of pgg or pa treatment was lower than those in the group without the treatment with the test compounds. mrna expression of ldlr in mrc was likewise assessed (fig b) . ldlr mrna expression in the hrv- or hrv- infected untreated cells was similar to that in the mock untreated cells and the expression decreased in the group of pa or pgg treatment. using elisa (fig c) , it was found that sicam- levels in supernatants of hrv- or hrv- infected cultures were higher than those in supernatants of noninfected cultures, and sicam- levels in supernatants of cultures treated with pa or pgg were significantly lower than those in cultures without the treatment with the test compounds. however, there was no difference in sicam- levels between mock cultures with or without constituents. western blot analysis of micam- and ldlr expressions in mrc cells days after hrv infection in the presence of μg/ml pa or μg/ml pgg was performed. micam- protein levels in noninfected cultures were similar to those in cultures infected with hrv- or hrv- , and the protein levels in cultures treated with pa or pgg at the test concentrations were also similar to those in cultures without treatment of the test compounds (fig a for hrv- ; fig b for hrv- ) . the presence of μg/ml pgg in cultures resulted in the sharp decline in the ldlr protein expression, compared with the cultures without pgg treatment, regardless of hrv- infection. however, μg/ml pa had no effect on expression of ldlr protein (fig c) . similar results were also observed with hrv- ( fig d) . effect on expression of cytokines mrna expressions of various cytokines in mrc days after infection with hrv- or hrv- in the presence of μg/ml pa or μg/ml pgg were assessed using real-time qrt-pcr. hrv- and hrv- evoked a significant increase in il- mrna expression levels in the nontreated cell cultures, but the expression levels were significantly reduced in the cell cultures treated with pa or pgg, although there was no difference in il- mrna levels between mock cultures with or without constituents (fig a) . similar results were also observed with il- mrna (fig b) . the expression of tnf mrna in the culture challenged with hrv- or hrv- was similar to that in the noninfected cell cultures. the treatment with pa and pgg did not remarkably reduce the tnf mrna expression, compared to the untreated cells ( fig c) . although the ifn-β mrna expression did not occur in the noninfected cell cultures, ifn-β mrna was expressed in the cell cultures infected with hrv- or hrv- . the ifn-β mrna expressions were meaningfully inhibited by pa or pgg treatment (fig d) . il- β mrna expression was also highly induced by hrv- or hrv- , and the induction disappeared or was significantly reduced by pa or pgg treatment (fig e) . the concentrations of il- detected by elisa in the hrv- or hrv- infected cell supernatants were significantly higher than those in the noninfected cell supernatants. il- concentrations in the mock cultures treated with pa were higher than those in the mock cultures without the treatment. however, il- concentrations in infected cultures were reduced by treatment with μg/ml pa or μg/ml pgg. the decrease in hrv-induced il- secretion by μg/ml pgg treatment was higher than that by μg/ml pa treatment. pa and pgg affected il- protein expressions in a concentration-dependent manner (fig a) . similar results were observed with il- ( fig b) . however, tnf protein was not detectable in supernatants of all the cultures (data not shown). effect on tlr mrna expression tlr mrna expression in hrv- or hrv- infected cells was increased, compared with uninfected cells (fig ) . tlr mrna expression in the cultures treated with μg/ml pa or μg/ml pgg was significantly lower than that in the cultures without the treatments with the test compounds. many plants and their constituents such as phenolics, terpenoids and alkaloids manifest antiviral activity toward different viruses [ , ] and have been proposed as alternatives to conventional antiviral drugs. anti-hrv constituents derived from plants include alkaloids (e.g., arborinine and (s)-ribalinine, ic . and . μm [ ] ; glaucine, ic doi: . /journal.pone. .g [ ] ; gallic acid, ic . μm [ ] ). it has been reported that hrv capsid-binding compounds toward all hrv serotypes showed the existence of group a and b, based on a wide range of susceptibilities to antiviral compounds [ ] . in the current study, the antiviral principles of p. lactiflora root was determined to be the aryl ketone pa ( ), the simple benzoic acid ga ( ) , and the gluco-hexose pgg ( ). these constituents exhibited antiviral activity toward both group a (hrv- ) and group b (hrv- ). ic of pgg, ga, and pa toward two hrvs was between . and . μm, between . and . μm, and between . and . μm, respectively, although ic of the natural compounds stated previously is between . and . μm. pgg exhibited greater antiviral activity than ribavirin (ic , . - . μm) toward two hrvs and high selectivity. the virus titration assay results also proved that the constituents had antiviral activity toward the hrvs. this original finding indicates that materials derived from p. lactiflora root can hold promise for the development of novel and effective naturally occurring antiviral agents for two different hrv groups a and b. pgg was reported to possess antiviral activity toward hepatitis b virus [ ] and influenza a virus [ ] . investigations on the modes of action of natural antivirals may contribute to the development of selective hrv therapeutic alternatives with novel target sites. the modes of antiviral action of plant secondary substances have been well documented by rollinger and schmidtke [ ] and kitazato et al. [ ] . targeting virus molecules is likely more specific and less toxic, but there is a narrow spectrum of viruses and a higher risk of creating resistant viruses [ ] . on the contrary, chemicals which target cellular molecules may possess a broader antiviral activity spectrum and less risk of developing virus resistance, but may be more toxic to the host cell [ ] . in the current study, pgg and pa do not interact with the hrv- particles, as preexposure of the virus to the constituents did not alter the infectivity of hrv- particles. based on time-course tests, pgg and pa can inhibit hrv- infection only when they were added during the virus inoculation ( h), but not after h or later. this finding suggests that pgg and pa may interact with the human cells in the early stage of hrv infections to protect the cells from the virus destruction. the mechanism of action of these constituents on cellular protection toward hrv- still remains unclear, although ribavirin was reported to enter host cells through nucleoside transporters in several studies [ , ] . in addition, real-time rt-pcr analysis revealed that the rna replication levels of hrvs were remarkably reduced in the mrc cultures treated with these constituents. this finding suggests that pgg and pa may block or reduce the entry of the viruses into the cells in the early stage of hrv infections to protect the cells from the virus destruction and abate virus replication. detailed tests are needed to fully understand the modes of action of the constituents. it has been suggested that the mode of anti-hrv action of gallic acid might be derived from the inhibition of virus absorption [ ] . interference with specific virus-host cell receptor interactions can be one of the potential interventions in antiviral chemotherapy [ ] . hrvs are classified into the major and minor groups, based on their binding to icam- [ , ] or to members of the ldlr family [ ] , respectively. icam- is a critical target-docking molecule on epithelial cells for % of the hrv serotypes [ , ] . the major group uses icam- as a mechanism to gain entry to the host cell [ ] . it has been suggested that antagonism of the virus-receptor interaction would appear to be an effective way to inhibit a broad spectrum of hrvs [ ] . macrolide antibiotics such as erythromycin inhibit infection by the major group of hrvs via a reduction in icam- expression [ ] . micam- expression is equally induced by both major and minor group hrvs [ ] . the hrv-induced icam- up-regulation is not receptor-restricted [ ] . moreover, it has been reported that pretreatment of hrv- with sicam did not alter the ability of hrv- to induce icam- [ ] . the current experiments demonstrated that both hrv- and hrv- induced the both forms of icam- expressed in mrc . elisa results revealed that sicam- protein levels of infected cultures treated with pa or pgg were significantly lower than those in cultures without the compound treatment, although there was no difference in sicam- levels between mock cultures with or without the constituents. however, icam- mrna expression levels were decreased in the presence of pgg or pa in both infected and noninfected cell cultures. the findings indicated that these constituents interfered with host cell protein expression of this receptor through inhibition of the receptor rna expression or reduction of hrv replication. in addition, ldlr mrna expressions were also reduced in the presence of pgg and pa in the cultures. however, ldlr protein expression was inhibited in pgg treatment only. the findings suggest that pgg interfere with ldlr expression through inhibiting expression of ldlr rna or reduction of hrv replication, whereas pa interfered with ldlr expression through only reduction of hrv replication. pa was reported to suppress icam- expression in tnf-stimulated human umbilical vein endothelial cells [ ] . hrv infection induces the production of numerous components of innate immunity including tlr and imflamatory mediators, such as kinins, leukotrienes, histamine, interleukins (il- , il- , and il- ), tnf, and regulated by activation normal t cell expressed and secreted [ , ] . the surface-expressed tlr s were reported to have an important function in response to a common human viral infection of natural host cells and play an important role in innate immune responses toward hrv infection [ , ] . during the replication cycle of hrv, viral double-stranded rna (dsrna) is recognized by tlr [ ] . the interaction between dsrna and tlr activates a signaling cascade that triggers increase of cytokine production in host cells and stimulates innate immune responses [ ] . it was supposed that inhibition of tlr expression have potential modulatory effects on the pathophysiologic changes related to hrv infection [ ] . cytokines such as tnf, il- β, il- , and il- induce upregulation expression of the adhesion molecules such as icam- in both epithelial and vascular endothelial cells, thereby increasing cell susceptibility to hrv infection [ ] . the concentrations of il- and il- in nasal secretions correlate with the severity of the symptoms in patients with colds [ ] . hrv infections were reported to be responsible for triggering exacerbations of asthma through inducing gene expression of these cytokines in asthmatic subjects [ ] . elisa results revealed that pa induced il- and il- expression. therefore, the expression levels in the infected cell cultures treated with pa were lower than those in the infected cell cultures without the constituent as a consequence of the hrv replication reductions. pgg reduced il- and il- expression and the decreases of expression levels in the infected cell cultures treated with pgg were resulted from the interference of the constituent with both of host cell protein expression and hrv replication. the current study also indicated that the constituents reduced expressions of tlr and cytokines, such as tnf, ifn-β, and il- β, which resulted in diminishing symptoms induced by hrv. it may be that effects of the constituents on blocking or reducing the entry of the viruses into the cells, which results in reducing hrv-rna replications, play an important role in interfering with hrv-induced gene expression. in conclusion, p. lactiflora root-derived preparations containing pgg and pa could be useful as an antiviral agent in the prevention or treatment of hrv infection. the antiviral action of pgg and pa may be an indication of at least one of the pharmacological actions of p. lactiflora. for the practical use of the preparations as novel anti-hrv products to proceed, further research is needed to establish their safety with respect to humans and whether this activity is exerted in vivo after consumption of p. lactiflora root-derived products by humans. lastly, detailed tests are needed to understand how to improve anti-hrv potency and stability for eventual commercial development. the human rhinovirus: human pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery human rhinoviruses detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr respiratory infections: a current and future threat job satisfaction and short sickness absence due to the common cold excessive antibiotic use for acute respiratory infections in the united states the treatment of rhinovirus infections: progress and potential can ethnopharmacology contribute to the development of antiviral drugs? review of antiviral and immunomodulating properties of plants of the peruvian rainforest with a particular emphasis on una de gato and sangre de grado plants and human health in the twenty-first century novel antiviral agents: a medicinal plant perspective chinese drugs of plant origin mechanisms of action of ribavirin against distinct viruses tetrazolium (mtt) assay for cellular viability and acidity growth-inhibiting, bactericidal, and urease inhibitory effects of paeonia lactiflora root constituents and related compounds on antibiotic-susceptible and-resistant strains of helicobacter pylori isothiazole derivatives as antiviral agents two phloroglucinol glucosides, flavan gallates and flavonol glycosides from sedum sediforme flowers the isolation of , , , , -penta-o-galloyl-beta-d-glucose from acer truncatum bunge by high-speed counter-current chromatography in vitro antiviral activities of caesalpinia pulcherrima and its related flavonoids a colorimetric-based accurate method for the determination of enterovirus titer antiviral activity of quercetin -rhamnoside against porcine epidemic diarrhea virus cleavage of structural proteins during the assembly of the head of bacteriophage t . nature antiviral properties of phytochemicals in silico target fishing for rationalized ligand discovery exemplified on constituents of ruta graveolens cinnamoyl-and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities structure-based virtual screening for the discovery of natural inhibitors for human rhinovirus coat protein antipicornavirus flavone ro - plant antiviral agents v. -methoxyflavones as potent inhibitors of viral-induced block of cell synthesis antiviral activity of natural occurring flavonoids in vitro constituents of eriobotrya japonica: a study of their antiviral properties inhibitorty effects of orobol -o-d-glucoside from banaba (lagerstroemia speciosa l.) on human rhiniviruses replication antiviral activity of raoulic acid from raoulia australis against picornaviruses anti-human rhinovirus activity of gallic acid possessing antioxidant capacity two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity in vitro antiviral activity of , , , , -penta-o-galloyl-beta-d-glucose against hepatitis b virus antiviral activity and possible mechanisms of action of pentagalloylglucose (pgg) against influenza a virus viral infectious disease and natural products with antiviral activity ribavirin uptake by human erythrocytes and the involvement of nitrobenzylthioinosine-sensitive (es)-nucleoside transporters reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance human rhinoviruses the major human rhinovirus receptor is icam- the major and minor group receptor families contain all but one human rhinovirus serotype members of the lowdensity lipoprotein receptor family mediate cell entry of a minor-group common cold virus rhinovirus infection induces expression of its own receptor intercellular adhesion molecule (icam- ) via increased nf-kappab-mediated transcription erythromycin inhibits rhinovirus infection in cultured human tracheal epithelial cells ifn-gamma regulation of icam- receptors in bronchial epithelial cells: soluble icam- release inhibits human rhinovirus infection paeonol suppresses intercellular adhesion molecule- expression in tumor necrosis factor-α-stimulated human umbilical vein endothelial cells by blocking p , erk and nuclear factor-κb signaling pathways cooperative effects of rhinovirus and tnf-alpha on airway epithelial cell chemokine expression toll-like receptor is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells levocetirizine inhibits rhinovirus-induced icam- and cytokine expression and viral replication in airway epithelial cells carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells association between interleukin- concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma key: cord- - x oh q authors: arruda, l. karla; solé, dirceu; naspitz, charles k. title: early interventions in allergic diseases date: journal: allergy frontiers: therapy and prevention doi: . / - - - - _ sha: doc_id: cord_uid: x oh q atopy has been defined as the genetic predisposition to develop ige antibody responses to a variety of common environmental allergens. clinically, atopy is expressed by asthma, allergic rhinoconjunctivitis and atopic dermatitis. it has been recognized that the “atopic march” evolves from food allergy and atopic dermatitis in the first years of life, followed by asthma and allergic rhinitis. over the past years, the prevalence of allergies and asthma has increased significantly in developed countries, and asthma is one of the most common chronic diseases in children. evidence indicates that environmental factors acting early in life, including respiratory viral infections, exposure to pets and microbial products, day-care attendance, breast feeding, and exposure to allergens, tobacco smoke and other pollutants, are key events for establishment of sensitization and development of chronic, persistent symptoms of allergic diseases [ ]. it is thought that gene—environment interactions play a crucial role in these processes. therefore, attempts to successfully prevent development of allergic diseases should be a priority. at present, there are no genetic markers for atopy or asthma which could be used routinely in clinical practice and family history of atopy has been used to identify children genetically at-risk of developing allergic diseases. these children from high-risk families have been the focus of most of the intervention studies. atopy has been defined as the genetic predisposition to develop ige antibody responses to a variety of common environmental allergens. clinically, atopy is expressed by asthma, allergic rhinoconjunctivitis and atopic dermatitis. it has been recognized that the "atopic march" evolves from food allergy and atopic dermatitis in the first years of life, followed by asthma and allergic rhinitis. over the past years, the prevalence of allergies and asthma has increased significantly in developed countries, and asthma is one of the most common chronic diseases in children. evidence indicates that environmental factors acting early in life, including respiratory viral infections, exposure to pets and microbial products, day-care attendance, breast feeding, and exposure to allergens, tobacco smoke and other pollutants, are key events for establishment of sensitization and development of chronic, persistent symptoms of allergic diseases [ ] . it is thought that gene-environment interactions play a crucial role in these processes. therefore, attempts to successfully prevent development of allergic diseases should be a priority. at present, there are no genetic markers for atopy or asthma which could be used routinely in clinical practice and family history of atopy has been used to identify children genetically at-risk of developing allergic diseases. these children from high-risk families have been the focus of most of the intervention studies. in this chapter, we discuss risk factors for development of sensitization and allergic disease, focussing on preventive strategies for allergies and asthma at an early age. infections with respiratory viruses, particularly human rhinovirus (hrv) and respiratory syncytial virus (rsv) are leading causes of lower respiratory tract (ltr) illnesses associated with wheezing in children. although acute wheezing episodes may be severe enough to require hospitalization, majority of children presenting wheezing illnesses early in life will no longer wheeze by the age of [ ] . however, in a proportion of these children, early-life-wheezing is a clinical manifestation of asthma. respiratory viral infections have been implicated in the pathogenesis of asthma in several ways: during infancy, certain viruses have been linked to inception of the asthma phenotype; in children with established asthma, viral respiratory infections play a significant role in triggering acute exacerbations that might lead to hospitalizations and frequent outpatient visits; in children with repeated infections due to day-care attendance or contact with older siblings, respiratory viruses may have a paradoxical effect of reducing long-term risk of allergy and asthma, through alterations of cytokine response profiles. in the first years of life, most lrt illnesses with wheezing are associated with infection by rsv. in the northern hemisphere, rsv accounts for - % of wheezing episodes in children younger than years of age [ ] . it has been shown that children who wheeze with rsv infection in early life have lower level of lung function prior to infection [ ] . most children have serum rsv antibody by the age of , yet reinfections are common. although risk of subsequent wheezing after rsv may decrease significantly with age, recurrent episodes of wheezing due to active rsv infections may occur throughout childhood. transmission requires close contact, and occurs either by large-particle aerosols or by contamination of hands and inoculation into the eye or nose, with an average incubation period of - days [ ] . more recently, the role of hrvs in causing acute wheezing has been appreciated. hrvs are small, nonenveloped, positive-strand rna viruses in the family picornaviridae , with over identified serotypes with minimal cross-antigenicity [ ] . hrv infects only higher primates, and causes illness only in humans, with replication restricted to the respiratory epithelium [ ] . in temperate climates, hrv has been estimated to cause up to % of autumn colds [ , ] . in tropical countries, available evidence indicates that hrv is frequently associated with acute respiratory illnesses (ari). hrv transmission requires close exposure and occurs mainly by hand-to-hand contact, followed by self-inoculation into the eye or nose. it can also be transmitted by airborne spread. once hrv reaches the nasal cavity, infection occurs in virtually % of susceptible subjects; and approximately % of those infected develop illness after - days incubation [ ] . sensitive pcr-based assays have established the importance of hrv as the cause of lrt illnesses, in addition to upper respiratory tract symptoms. a recent study with in situ hybridization applied to lower airway biopsy specimens has demonstrated presence of hrv in the ltr of % of a group of children - months of age with recurrent respiratory symptoms [ ] . other viruses have also been associated with wheezing lrt illnesses in children at lower frequencies, including influenza, human parainfluenza viruses, human coronavirus, adenovirus, human metapneumovirus, and the recently identified human bocaviruses (hbov) -however at a lower frequency [ ] . in keeping with observations made in temperate climates, it has been shown that infection with respiratory viruses and family history of allergy, were independently associated with wheezing among infants [ ] . results of this case-control study carried out in ribeirão preto, a city in southeast brazil, revealed that, in the group of children under years of age, respiratory viruses were detected in . % of wheezing infants versus . % of controls, and rsv was detected in % wheezing children and none of the controls. rhinovirus rna was found in . and % of the wheezing and control children, respectively, though this difference was not significant ( p = . ). the frequency of rsv was lower than that reported in temperate regions ( % versus - %). however, considering the subgroup of infants - months-old, % tested positive for rsv antigen. rsv infections were predominantly found in the months of february to may, corresponding to late summer and early to midfall, indicating that the virus occurs in a different seasonal pattern as compared to that of the northern hemisphere. in the group of children - years of age, respiratory viruses were not significantly associated with wheezing [ ] . in the united states, heymann et al have shown that viral infections, especially hrv, were the dominant risk factor for wheezing among children hospitalized before the age of [ ] . in their study, % of wheezing children p = years-old were positive for virus, compared to % of controls ( p < . ); rsv was the dominant pathogen in the winter months among children years-old or younger; however, rhinovirus was detected more often among wheezing children hospitalized in the other months of the year ( %) as compared to controls ( %, p < . ) [ ] . one important issue would be whether infections with respiratory viruses particularly rsv and hrv occurring early in life could function as triggers or "adjuvants" for subsequent development of sensitization and persistent symptoms of allergic diseases. the rational for this hypothesis would be the potential of these infections to induce significant damage to the airways which might facilitate penetration of allergen(s) and/or trigger events related to airway remodeling. rsv enters the cell by fusion of the viral envelope with the cell membrane, and causes syncytia formation as a result of fusion of the infected cells to adjacent ones. replication in the bronchiolar epithelium causes necrosis of ciliated cells, peribronchiolar inflammation with abundant lymphocytes and macrophages, and impairment of secretion clearance, resulting in small airway obstruction and the hyperinflation characteristic of bronchiolitis. clinically, involvement of the lrt is characterized by tachypnea, dyspnea, cough, expiratory wheezing, air trapping, hyperaeration of the lungs on chest x-rays, and intercostal muscle retractions and cyanosis [ ] .the pathogenesis of hrv infection is based on the release of cytokines, chemokines, and inflammatory mediators triggered by productive viral replication in a limited number of cells. a number of chemokines, particularly cxcl (il- ), ccl (macrophage inflammatory protein a ) and ccl (rantes) are major mediators released during respiratory viral infections, which could recruit virus-specific t-cells as well as allergen-specific t-cells that in turn could augment any ongoing allergic response in the lung [ , ] . it has been speculated that the contemporaneous occurrence of cycles of viral-induced and allergen-induced inflammation in the airways during the period of rapid lung growth and remodeling in infancy interacts synergistically to disrupt underlying tissue differentiation programs. this interaction could result in deleterious changes in ensuing respiratory functions, which may then manifest as persistent wheeze and/or asthma [ ] . studies have shown association of rsv bronchiolitis and other early respiratory tract infections with recurrent wheezing or symptomatic asthma during the first - years of life [ , ] . a long-term study carried out in tucson, arizona, revealed an association of ltr infection caused by rsv early in life with persistent wheezing at and years of age; however, this effect was lost at age [ , ] . besides rsv, hrv [ ] may be potentially implicated in the subsequent development of childhood asthma. lemanske et al. have shown that, in a group of children at high risk of asthma, studied during the first years of life, infection with hrv in the first year was the greatest risk factor for persistent wheezing in the third year [ ] . the authors showed that % of children who wheezed during rhinovirus season continued to wheeze in the third year, as compared to only % of all other infants (or = . ). a study in finland revealed that infants hospitalized for rhinovirus-induced wheezing presented a fourfold higher risk of asthma in school age, as compared to wheezing infants from whom no rhinovirus was identified. children with atopic dermatitis were especially likely to develop wheezing during hrv infections [ ] . these studies highlight the previously unrecognized potential role of rhinovirus infection occurring in early life in the onset of asthma. follow-up of children - years of age who participated in the emergency room study in brazil [ ] revealed that, after years, % presented persistent wheezing. in contrast to studies carried out in temperate regions, viral infections were not a risk factor for persistent wheezing. on the other hand, early sensitization particularly to mites and cockroach, at - years of age, and exposure to high levels of cockroach allergen in the home in the first years of life were both strong and independent risk factors for persistence of wheezing. it has been consistently shown that early allergen sensitization becomes a major risk factor for wheezing exacerbations and hospitalizations for wheezing after age [ , [ ] [ ] [ ] [ ] . it is thought that ige-mediated inflammation found in most children with persistent symptoms of asthma is a key factor in causing lung function impairment and airway remodeling. previous studies in brazil have shown that day-care centers and schools, in addition to homes, are sources of significant exposure to mite and cockroach allergens, which might contribute to sensitization [ , ] . heymann et al have demonstrated that sensitization to house dust mites and other aeroallergens was an important risk factor for hospital admissions for wheezing and adverse responses to viral infections, particularly those caused by rhinovirus, in - years old children [ ] , highlighting the synergistic effect of sensitization, allergen exposure, and concomitant viral infection in augmenting inflammatory responses in the airways [ ] . the possibility that viral and atopy-associated inflammation may interact synergistically to drive asthma pathogenesis has been raised recently by kusel et al. [ ] . results of this community-based cohort, involving children followed from birth to years, revealed that acute ltr infection caused by rhinovirus or rsv in the first year of life interacted with atopy in infancy (sensitization £ years-old) to promote later asthma [ ] . it is well recognized that exacerbations of asthma, in patients with established disease, are often triggered by respiratory viral infections, particularly those caused by rhinovirus. in asthmatic patients, persistence of hrv up to weeks following infection or exacerbation of asthma, has been reported [ , ] , suggesting that an aberrant immune response to hrv may be involved in the development of acute exacerbations in atopic individuals with asthma. also, coexistence of atopy enhances the clinical effect of hrv infection, increasing intensity and duration of bronchial hyperreactivity [ ] . finally, it has been suggested that repetitive viral infections might confer protection to development of asthma, based on their ability to skew the immune system away from the th -type response [ , ] . day-care attendance and/or siblings significantly increased the likelihood of occurrence of rsv or rhinovirus infections, and increased the risk of rhinovirus-induced wheezing at an early age. neonatal interferon (ifn)-g responses were lower in infants with high frequency of respiratory infections; conversely, frequent infections were associated with a smaller decline of ifn-g responses during the first year of life, indicating that preexisting immunologic factors may influence the expression of viral infections in infancy [ ] . exclusive breast feeding for at least months has been associated with protection against development of asthma or atopic diseases [ , ] , but other studies have failed to demonstrate protection by breast milk [ ] . bottcher et al. [ ] found no relationship in levels of cytokines (il- , il- , il- , il- , il- , il- , il- , ifn-g , tgf-b , tgf-b ), chemokines (rantes, eotaxin) or secretory iga in breast milk, and development of sensitization or allergic symptoms, or levels of salivary iga during the first years of life. endotoxin is a constituent of the outer membrane of gram-negative bacteria, found ubiquitously in nature, being present in most indoor environments as a component of house dust. endotoxin stimulates the release of potent proinflammatory cytokines. exposure to high levels of endotoxin in dust is associated with induction of asthma in sensitive patients [ , ] . it has been demonstrated that bacterial endotoxin is capable of producing th associated cytokines, ifn-g , and il- and therefore, has the potential to decrease allergen sensitization. chronic endotoxin exposure, before polarized t-cell responses are established, might be expected to protect against allergen sensitization by continuously enhancing th -type lymphocyte development [ ] . this assumption has been partially confirmed by studies in humans showing that exposure to high levels of endotoxin in early life was associated with protection against allergic sensitization [ , ] . an experimental study with pregnant balb/c mice has shown that combined exposure to endotoxin during prenatal and postnatal phases suppressed allergenspecific sensitization (ige production), eosinophilic airway inflammation (reduced numbers of eosinophils in bronchoalveolar lavage fluids), and in vivo airway reactivity in response to methacholine. the suppression of allergen-mediated inflammatory responses was associated with increased toll-like receptor and t-bet expression by lung tissues and a shift toward predominantly th immune responses [ ] . similar results were observed by wang and mccusker [ ] . the relationship of exposure to microbial agents (endotoxin, fungal agents, and other microbial contaminants) early in life ( months of age) and the development of atopic sensitization and physician-diagnosed asthma and wheeze in the first years of life, in children of atopic mothers, was investigated in the prevention and incidence of asthma and mite allergy (piama) birth cohort study. a significant reduction in the development of asthma was associated with early exposure to these substances [ ] . children who were born and raised in a farm environment and exposed to poultry and livestock were reported to have lower prevalence of asthma and/or allergic diseases in comparison to those living in urban area [ , ] . until recently, exposure to high levels of endotoxin was associated with exposure to farm animals, presence of pets in home, number of people living in the house, and cleaning habits [ ] . however, results of a study carried out on children from rural areas in europe, evaluating farm-related exposures and health outcomes, revealed that levels of endotoxin and extracellular polysaccharides were associated with health outcomes independent of farm exposures [ ] . it has recently been shown by simpson et al. that the impact of endotoxin may be genetically determined [ ] . in the setting of a birth cohort study, increasing endotoxin exposure was associated with reduced risk of allergic sensitization and eczema, and increased risk of nonatopic wheeze, only in children with the cc genotype at − of the cd gene [ ] . several prospective birth cohort studies have raised the issue of whether keeping of pets , particularly keeping of dogs or cats, might decrease the risk of developing sensitization to those allergens and have confirm these results in part [ ] [ ] [ ] . a systematic review of the scientific literature concerning keeping of pets within the first years of life and prevalence of asthma has shown that exposure to pets was associated with increased risk of asthma and wheezing in children older than years of age and a tendency for protection in those aged below years [ ] . in a recent study, a protective effect of early exposure to cat was documented [ ] ; however it hasn't happened unanimously and some bias of selection may have accounted for the results. so, the protective effect observed might be attributable to allergen or other exposures associated with pet ownership (eg. endotoxin), but may in part be due to the prior removal of pets in families where children are sensitized or symptomatic or in families with a positive history for atopy at the time the child was born [ ] . platts-mills and colleagues [ ] while evaluating the immune response among children, of whom had asthma and airway hyperresponsiveness, have demonstrated that increasing the exposure to house dust mites was associated with an increase in frequency of sensitization to dust mite allergen. the highest category of exposure to cat allergen though was associated with decreased frequency of sensitization and higher prevalence of igg antibody to fel d . however, the occurrence of sensitization to dust mite or cat allergens was the strongest independent risk factor for asthma (mite or = . ; cat or = . ). maternal tobacco smoking during gestation is an important avoidable risk factor associated with elevated levels of ige in cord blood, subsequent asthma and allergic diseases in childhood [ ] [ ] [ ] , and reduction of pulmonary function in children [ ] . increased production of il- by cord blood cells has been found in newborns whose mothers had smoked during gestation as compared with those who never smoked [ ] . macaubas et al. [ ] reported a direct relationship of maternal tobacco smoking with both low concentrations of il- and ifn-g in cord blood and increased risk of wheezing by age years. a recent experimental study on balb/c mice has shown that daily in utero exposure to maternal tobacco smoking was associated with exacerbation of subsequent adult responses to initial allergen exposure [ ] . there is a strong body of evidence to support the role of exposure of children to environmental tobacco smoke (ets) in increasing the incidence of asthma, wheeze, cough, bronchitis, bronchiolitis, pneumonia, and impaired pulmonary function. ets increases both the prevalence and severity of asthma, as judged by increases in the frequency of attacks, the number of emergency room visits, and the risk of intubation [ , ] . the risk associated with parental smoking seems to be greater at younger ages. although a dose-response relationship between ets exposure and respiratory outcomes has been demonstrated, at present there is no threshold dose of ets exposure below which an effect will not occur, and therefore active intervention measures and policies to reduce or eliminate children's exposure to ets should be strongly encouraged [ ] . polymorphisms in the proinflammatory cytokine genes tumor necrosis factor-a (tnf) and lymphotoxin-a (lta) have been associated with asthma and atopy in some studies. secondhand smoke and ozone both stimulate tnf production. in a recent study, wu et al. genotyping six tagging single nucleotide polymorphisms (snps) in tnf and lta have observed that genetic variation in tnf may contribute to childhood asthma and that association may be modified by parental smoking [ ] . a home-based, individualized, intervention study [ ] carried out among inner city children with atopic asthma which included education and remediation for exposure to both allergens and ets, resulted in reduction of asthma associated morbidity. few specific interventions are available to reduce the impact of respiratory viruses. no vaccine is currently available for rsv prophylaxis. the disease enhancement caused by formalin-inactivated vaccine in the s plus results of more recent unsuccessful trials of live-attenuated vaccines, have significantly slowed progress toward an rsv vaccine. passive immunization/immunoprophylaxis with monthly infusions of rsv immunoglobulin or monthly intramuscular injections of humanized monoclonal antibody, during the rsv season, reduced the incidence and severity of rsv infections in high-risk children including those preterm babies less than months-old, children with congenital heart disease, and children less than years with bronchopulmonary dysplasia [ ] . the large number of hrv serotypes with minimal cross-antigenicity has hampered the development of an hrv vaccine. it may be possible to reduce exposure to hrv by washing of hands after contact with a cold sufferer or after handling objects that may have been contaminated with respiratory secretions [ ] . immunization with formalin-inactivated or live-attenuated multivalent influenza virus vaccines and chemoprophylaxis for influenza virus a are the methods available for preventing influenza. influenza vaccine is used prior to the influenza season. the inactivated vaccine has an approximate - % efficacy in preventing illness in healthy children and adults. in summary, there are virtually no effective strategies targeted at the respiratory viruses for treatment or prevention of viral-induced wheezing illnesses in children. however, evidence indicates that treatment of lung inflammation with inhaled corticosteroids or blocking viral-induced overproduction of leukotrienes with leukotriene-receptor antagonist montelukast may be effective in decreasing severity and frequency of viral-induced wheezing in young children with recurrent or persistent symptoms [ , ] . a double-blind, controlled trial (previa study) investigated the effect of treatment with montelukast for months in - years-old children with intermittent asthma. approximately half of these children were positive for at least one respiratory virus, including hrv, coronavirus, and rsv in their nasal aspirates during exacerbations of symptoms. the results showed that montelukast had a beneficial effect, decreasing frequency of exacerbations, increasing time between acute wheezing episodes, and reducing the need for inhaled corticosteroids during exacerbations [ ] . preventive strategies have focused on manipulating the environment of high-risk individuals as an attempt to reduce the prevalence of allergies and asthma in children. at present, six primary prevention controlled studies are in progress [ ] . the longest follow-up reported has been from the isle of wight study [ ] . in this randomized, controlled study, a group of high-risk infants was recruited prenatally, and development of allergic diseases and sensitization to common allergens was assessed at ages , , and years. intervention included strict elimination of common food allergens (dairy products, egg, wheat, nuts, fish, and soy) to the age of months. lactating mothers followed the same restriction diet (except wheat) for the duration of breast feeding. extensively hydrolyzed formula was given as a supplement to the child from birth, or when breast feeding was discontinued before months. stringent allergen avoidance measures were also instituted at birth, aimed at reducing exposure to house dust mites. repeated measurement analysis showed a sustained preventive effect of allergen avoidance on asthma, atopic dermatitis, and sensitization to allergens over the period of the first years of life, and on allergic rhinitis at age [ ] . therefore, the conclusion was that stringent avoidance of mite and food allergens applied to high-risk children in infancy were beneficial and resulted in reduction of allergic sensitization and clinical manifestations of allergy, beyond the period of avoidance [ ] . likewise, outcome of the canadian primary prevention study on high risk infants has been reported at age years, showing that intervention during the first year of life, comprising avoidance of mite, pet allergens and ets, as well as dietary regimen, resulted in reduction of asthma symptoms and asthma diagnosed by a pediatric allergist in the intervention group. in the canadian study, no significant effect of intervention was observed for bronchial hyperreactivity, allergic sensitization, allergic rhinitis, or atopic dermatitis at age years [ ] . initial results from other cohorts look promising; however further follow-up will be necessary before any recommendations can be made [ ] . results of the manchester asthma and allergy study (maas) have been reported up to the age of , and showed that stringent mite and pet allergen avoidance measures starting during gestation, resulted in decrease in severe wheezing and exercise induced wheezing at age , and improved pulmonary function in the intervention group at age . however sensitization to mites was increased at years of age [ ] . in the study of prevention of allergy in children in europe (space), environmental control measures aimed at reducing exposure to dust mite allergens at birth and education failed to prevent sensitization at age [ ] . another study looking at the effects of mite avoidance measures during gestation and education. the piama study, showed a modest benefit of reduction of cough apart from colds at years of age [ ] . the results of the childhood asthma prevention study (caps), carried out in australia, revealed that house dust mite allergen avoidance in conjunction with supplementation of diet with omega- fatty acids (abundant in fish and canola-based oils), applied to children with high risk of asthma, resulted in decrease in cough and sensitization to mites at years of age , with no significant differences in wheeze [ ] . the conclusion of these prospective studies so far is that environmental measures taken to decrease exposure to dust mite allergens, even if started during gestation, appear to have limited beneficial effects. however, dust mite avoidance in conjunction with stringent dietary avoidance measures applied to high-risk infants of highly motivated families, may result in prevention of sensitization and clinical manifestations of allergy up to years. follow-up of some of the studies is still too short to allow more definitive recommendations. the prophylactic treatment with an antihistamine, ketotifen, in atopic dermatitis patients was followed by a fourfold reduction in incidence of asthma related symptoms, mainly in those with high levels of serum total ige [ ] . similar results were observed among children with high risk of developing asthma. the incidence of asthma in preasthmatic patients treated with ketotifen was % versus % in the placebo group [ ] . warner et al. have evaluated long-term treatment with cetirizine as a preventive tool for the onset of asthma in children aged less than years with atopic dermatitis and without asthma, in a double-blind, randomized, placebo-controlled trial (the early treatment of the atopic child, etac study). at the end of months of active treatment, they observed a significant reduction in the onset of asthma among grass pollen-sensitized infants and dust mite-sensitized infants. these differences were sustained only for the grass pollen-sensitized infants after months of treatment interruption. they concluded that cetirizine truly delays or, in some cases, prevents the development of asthma in a subgroup of infants with atopic dermatitis sensitized to grass pollen and, to a lesser extent, to house dust mite [ ] . more recently, preliminary results of the early prevention of asthma in atopic children (epaac) study, shows that the use of levocetirizine daily for months was safe among atopic children - months of age [ ] . on the whole, these studies indicate that the antihistamines ketotifen, cetirizine, and levocetirizine are safe for use in very young atopic children, and that they may have a role in preventing development of asthma in some of these children, particularly those with atopic dermatitis, and those allergic to house dust mite and grass pollen at an early age [ ] . the hypothesis that early introduction of inhaled corticosteroids in young children at high risk of developing asthma could change the natural history of the disease has been investigated. a recent study in preschool children at high risk of asthma revealed that years of treatment with inhaled corticosteroid was highly effective in reducing symptoms and asthma exacerbations, though the benefit was no longer present during a third treatment-free year, indicating that corticosteroids may not have disease modifying effects [ ] . in this trial, the prevention of early asthma in kids (peak), children aged - years were randomized to receive either mcg fluticasone twice daily for years or placebo, and at the end of the second year, treatments were interrupted. clinical and functional differences favoring the children treated with inhaled fluticasone disappeared a few weeks after discontinuation of regular treatment. recently, two other studies carried out in the united kingdom and denmark [ , ] reached similar conclusions as the peak trial: very early treatment of asthma with inhaled corticosteroids, even before the persistent form of the disease has become evident, does not change the natural clinical course of the disease, and does not seem to affect the level of lung function attained at the end of follow-up, despite the fact that this form of treatment is very effective in controlling asthma symptoms while in use [ ] . specific immunotherapy (sit) administered by the subcutaneous route is an efficient treatment for ige-mediated disease to defined allergens [ ] . beneficial effects of sit in children have been demonstrated in preventing new sensitizations in children monosensitized to mites [ , ] and in slowing the progression to asthma in those with seasonal allergic rhinitis, sensitized to pollen allergens (the pat study) [ ] . follow-up of children with allergic rhinitis sensitized to birch or grass pollens, who underwent sit for years [ ] showed that the effect of sit in preventing development of asthma was still evident years after sit was discontinued [ ] . concerns regarding the use of sit in asthma include the possibility of severe anaphylaxis; however guidelines have been developed to minimize risks of reaction [ ] . sublingual immunotherapy (slit) is increasingly being regarded as an efficient tool for the treatment of patients with asthma and/or rhinitis, as indicated by results of meta-analysis of studies carried out in children and adults [ ] . the increased safety and ease of administration of slit makes this strategy very attractive as a form of early intervention in young children with ige-mediated disorders, which could modify the natural course of allergic diseases. studies addressing the use of slit in young children however have not been reported. issues including standardization of the vaccines, establishment of effective doses and schedules for administration, compliance, and better understanding of mechanisms of action and magnitude of efficacy need further research [ ] . evidence suggests that events taking place between and years of age might be crucial determinants in the development of allergies and asthma [ ] . strategies to prevent development of sensitization and progression to disease or to elicit long lasting remission of symptoms are strongly desirable. however, current environmental interventions and treatment modalities with pharmacotherapy do not meet these expectations. according to the recent published world allergy organization project report and guidelines on prevention of allergy and allergic asthma document [ ] , some evidence-based recommendations can be highlighted (tables and ). acknowledgments dr. l. karla arruda's research on risk factors for asthma in children in brazil is supported by fapesp and cnpq, instituto de investigação em imunologia, l.k.a. and d.s. are recipients of cnpq scholarships. table recommendations based on the world allergy organization project report and guidelines on prevention of allergy and allergic asthma -primary prevention infants without a special risk for allergic diseases exclusive breast feeding for months is recommended by the who: if a supplement is needed, a conventional cow-milk-based formula is recommended (b) no special maternal diet during pregnancy or lactation (a) avoidance of solid foods until [ ] months of age (b) avoidance of exposure to tobacco smoke (also during pregnancy) (b) infants with a high risk for allergic diseases exclusive breast feeding for at least months: if a supplement is needed, a documented hypoallergenic formula is recommended for the first months of life; after the age of months, high-risk children can receive the same nutrition as nonhigh-risk children (a) no special maternal diet during pregnancy or lactation (a) avoidance of solid foods until [ ] months of age (b) environmental measures avoidance of tobacco smoke (also during pregnancy) (b) reduction of allergen exposure early in life (house dust mites, furred pets, cockroaches) (b) avoidance of damp housing conditions (c) avoidance of pollutants (c) table recommendations based on the world allergy organization project report and guidelines on prevention of allergy and allergic asthma -secondary prevention avoidance of tobacco smoke (b) patients who have perennial asthma, rhinitis, or eczema and who are allergic to house dust mites or animal dander should try to reduce their exposure to the relevant allergens (a, b). recommended measures include: removal of relevant pets reduction of indoor relative humidity below % if possible encasing of mattresses with documented protective coverings washing of pillows in hot water (> °c) regularly or encasing of pillows with documented protective coverings washing of bedding in hot water (> %) regularly (every - weeks) removal of carpets in bedroom influences in allergy: epidemiology and the environment development of wheezing disorders and asthma in preschool children the role of viral infections in the natural history of asthma respiratory syncytial virus and parainfluenza viruses clinical virology localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization frequency and natural history of rhinovirus infections in adults during autumn human rhinovirus in bronchial epithelium of infants with recurrent respiratory symptoms detection of viruses identified recently in children with acute wheezing risk factors for acute wheezing among children in a subtropical environment: role of respiratory viruses, ige antibodies and allergen exposure viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing respiratory viral infections drive chemokine expression and exacerbate the asthmatic response cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma viral infections and asthma inception respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age respiratory syncytial virus in early life and risk of wheeze and allergy by age years tucson children´s respiratory study: to present rhinovirus-induced wheezing in infancy -the first sign of childhood asthma? rhinovirus illnesses during infancy predict subsequent childhood wheezing rhinovirusassociated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis a prospective study of wheezing in young children: the independent effects of cockroach exposure, breast-feeding and allergic sensitization the relevance of allergen exposure to the development of asthma in childhood exposure to house-dust mite allergen (der p i) and the development of asthma in childhood. a prospective study relationship of indoor allergen exposure to skin test sensitivity in inner-city children with asthma exposure to indoor allergens in homes of patients with asthma and/or rhinitis in southeast brazil: effect of mattress and pillow covers on mite allergen levels daycare centers and schools as sources of exposure to mites, cockroach, and endotoxin in the city of são paulo, brazil synergism between allergens and viruses and risk of hospital admission with asthma: case-control study persistence of rhinovirus and enterovirus rna after acute respiratory illness in children persistence of rhinovirus rna after asthma exacerbations in children rhinoviruses in the pathogenesis of asthma: the bronchial epithelium as a major disease target breast-feeding, infant formulas, and the immune system a review of the effects of breastfeeding on respiratory infections, atopy and childhood asthma long-term relation between breastfeeding and development of atopy and asthma in children and young adults: a longitudinal study cytokine, chemokine and secretory iga levels in human milk in relation to atopic disease and iga production in infants domestic endotoxin exposure and clinical severity of asthma severity of asthma is related to endotoxin in house dust environmental influences on asthma and allergy relation between house-dust endotoxin exposure, type t-cell development, and allergen sensitization in infants at high risk of asthma metropolitan home living conditions associated with indoor endotoxin levels prenatal initiation of endotoxin airway exposure prevents subsequent allergen-induced sensitization and airway inflammation in mice neonatal exposure with lps and/or allergen prevents experimental allergic airway disease: development of tolerance using environmental antigens does early indoor microbial exposure reduce the risk of asthma? the prevention and incidence of asthma and mite allergy birth cohort study to endotoxin and its relation to asthma in school-age children determinants of endotoxin levels in living environments of farmers' children and their peers from rural areas surface sampling for endotoxin assessment using eletrostatic wiping cloths not all farming environments protect against the development of asthma and wheeze in children endotoxin exposure, cd , and allergic disease: an interaction between genes and the environment the pasture project: eu support for the improvement of knowledge about risk factors and preventive factors for atopy in europe childhood environmental and adult atopy: results from the european community respiratory health survey exposure to dogs and cats in the first year of life and risk of allergic sensitization at to years of age systematic review: exposure to pets and risk of asthma-like symptoms early" cat ownership and the risk of sensitization and allergic rhinitis in ligurian children with respiratory symptoms sensitisation, asthma, and a modified th response in children exposed to cat allergen: a population-based cross-sectional study prenatal and postnatal environmental tobacco exposure and children's health maternal smoking in pregnancy alters neonatal cytokine responses maternal smoking during pregnancy increases the risk of recurrent wheezing during the first years of life (bamse) effects of early onset asthma and in utero exposure to maternal smoking on childhood lung function association between antenatal cytokine production and the development of atopy and asthma at age years in utero exposure to environmental tobacco smoke potentiates adult responses to allergen in balb/c mice parental smoking modifies the relation between genetic variation in tumor necrosis factor-a ( tnf ) and childhood asthma inner-city asthma study group. results of a home-based environmental intervention among urban children with asthma long-term inhaled corticosteroids in preschool children at high risk for asthma montelukast reduces asthma exacerbations in -to -year-old children with intermittent asthma allergen avoidance in the primary prevention of asthma primary prevention of asthma and atopy during childhood by allergen avoidance in infancy: a randomised controlled study prevention of allergic disease during childhood by allergen avoidance: the isle of wight prevention study the canadian childhood asthma primary prevention study: outcomes at years of age custovic a; nac manchester asthma and allergy study group. early life environmental control: effect on symptoms, sensitization, and lung function at age years effect of mite-impermeable mattress encasings and an educational package on the development of allergies in a multinational randomized, controlled birth-cohort study - months results of the study of prevention of allergy in children in europe prevention and incidence of asthma and mite allergy (piama) study. placebo-controlled trial of house dust mite-impermeable mattress covers: effect on symptoms in early childhood three-year outcomes of dietary fatty acid modification and house dust mite reduction in the childhood asthma prevention study prevention of asthma by ketotifen in infants with atopic dermatitis prevention of asthma with ketotifen in preasthmatic children: a three-year follow-up study early treatment of the atopic child. a double-blinded, randomized, placebo-controlled trial of cetirizine in preventing the onset of asthma in children with atopic dermatitis: months' treatment and months' posttreatment follow-up early prevention of asthma in atopic children (epaac) study group. safety of levocetirizine treatment in young atopic children: an -month study custovic a; ifwin study team. secondary prevention of asthma by the use of inhaled fluticasone propionate in wheezy infants (ifwin): double-blind, randomized, controlled study intermittent inhaled corticosteroids in infants with episodic wheezing asthma treatment and asthma prevention: a tale of parallel pathways allergen immunotherapy: therapeutic vaccines for allergic diseases. world health organization. american academy of allergy immunotherapy with a standardized dermatophagoides pteronyssinus extrat. vi. specific immunotherapy prevents the onset of new sensitizations in children prevention of new sensitization in asthmatic children monosensitized to house dust mite by specific immunotherapy. a six year follow-up study pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the pat-study) five-year follow-up on the pat study: specific immunotherapy and long-term prevention of asthma in children sublingual immunotherapy: the optimism and the issues does environment mediate earlier onset of the persistent childhood asthma phenotype? preventive measures: early interventions key: cord- -gt pwc b authors: yang, albert c.; tsai, shih-jen; hong, chen-jee; wang, cynthia; chen, tai-jui; liou, ying-jay; peng, chung-kang title: clustering heart rate dynamics is associated with β-adrenergic receptor polymorphisms: analysis by information-based similarity index date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: gt pwc b background: genetic polymorphisms in the gene encoding the β-adrenergic receptors (β-ar) have a pivotal role in the functions of the autonomic nervous system. using heart rate variability (hrv) as an indicator of autonomic function, we present a bottom-up genotype–phenotype analysis to investigate the association between β-ar gene polymorphisms and heart rate dynamics. methods: a total of healthy han chinese adults ( males and females, aged . ± . years, range to years) were recruited and genotyped for three common β-ar polymorphisms: β( )-ar ser gly, β( )-ar arg gly and β( )-ar gln glu. each subject underwent two hours of electrocardiogram monitoring at rest. we applied an information-based similarity (ibs) index to measure the pairwise dissimilarity of heart rate dynamics among study subjects. results: with the aid of agglomerative hierarchical cluster analysis, we categorized subjects into major clusters, which were found to have significantly different distributions of β( )-ar arg gly genotype. furthermore, the non-randomness index, a nonlinear hrv measure derived from the ibs method, was significantly lower in arg homozygotes than in gly carriers. the non-randomness index was negatively correlated with parasympathetic-related hrv variables and positively correlated with those hrv indices reflecting a sympathovagal shift toward sympathetic activity. conclusions: we demonstrate a bottom-up categorization approach combining the ibs method and hierarchical cluster analysis to detect subgroups of subjects with hrv phenotypes associated with β-ar polymorphisms. our results provide evidence that β( )-ar polymorphisms are significantly associated with the acceleration/deceleration pattern of heart rate oscillation, reflecting the underlying mode of autonomic nervous system control. instantaneous heart rate in response to physiological perturbations often exhibits remarkable oscillations at multiple time scales. these oscillations, known as heart rate variability (hrv), are mainly mediated by the autonomic nervous system via parasympathetic and sympathetic innervations. analysis of hrv has been suggested to reveal subtle patterns of heart rate dynamics that are relevant to the underlying physiological state and autonomic nervous system function [ ] . prior studies have shown that hrv measures are highly heritable traits that can be used to support genetic association and linkage studies [ , , , ] . family and twin studies have shown a significant genetic influence on a variety of hrv measures [ , ] . genetic polymorphisms related to cardiovascular functions have been associated with altered hrv [ , , , ] . recently, we have also made progress in identifying variations in two genes related to neuropsychiatric function that are associated with altered heart rate dynamics in samples of healthy adult and elderly subjects: those encoding brain-derived neurotrophic factor (bdnf) [ ] and apolipoprotein e [ ] , respectively. despite increasing focus on investigating the genetic influence on autonomic functions, current approaches to genotype-hrv associations have largely been characterized by a top-down approach involving a direct comparison of continuous hrv variables among pre-defined groups of subjects (i.e., healthy vs. ill or groups of known genotypes), yet it is unclear how a particular genetic polymorphism may determine a similar pattern of autonomic heart rate control from one subject to another. specifically, heart rate dynamics is a phenotypic ''expression'' of the autonomic nervous system, so comparing similar heart rate oscillation phenotypes among individuals may reveal a global profile of autonomic function relevant to genetic variants. with these considerations in mind, in the present study, we introduce a bottom-up genotype-phenotype analysis to investigate the association between genetic polymorphisms and autonomic control of heart rate dynamics, using three common polymorphisms in genes encoding b-adrenergic receptor (b-ar) as an example. b-ar has a pivotal role in the functions of the cardiac autonomic nervous system. activation of b -ar provides strong stimulus to increase the frequency and contractility of the heart, whereas the activation of b -ar results in smooth muscle relaxation and increased cardiac output with less extent compared to b -ar. thus, the connection between b-ar and hrv is plausible and warrants evaluations. limited evidences suggest that variations in genes coding subtypes of b-ar may be associated with heart rate or hrv. for example, ser gly polymorphism in b -ar gene has been found to be associated with resting heart rate [ , ] , and an association of b -ar gene polymorphisms with spectral components of hrv measures has been reported in a relatively small healthy adult male sample [ ] . we applied an information-based similarity index (ibs) [ , ] to measure the pairwise dissimilarity of interbeat interval time series among a sample of healthy adult volunteers. the ibs method is based on rank-order frequency analysis of acceleration/ deceleration patterns of heart rate fluctuation. because stimulus of b-ar results in acceleration of heart rate, the functional changes in genetic polymorphisms of b-ar may affect acceleration/deceleration patterns of heart rate, which can be detected by the ibs method. the analyses of the present study were two-fold: ) a nonrandomness index [ ] derived from the ibs method was applied to quantify the nonlinear aspect of hrv according to b-ar genotype and to test the correlation of this index with standard hrv indices; and ) using agglomerative hierarchical cluster analysis, we unsupervisedly categorized these subjects into clusters based on pairwise dissimilarity among heart rate dynamics, and then we investigated the association of these clustering patterns with b-ar gene polymorphisms. we show that this bottom-up, categorization approach combining the ibs method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with b-ar gene polymorphisms. two hundred forty-seven healthy han chinese adult volunteers were recruited from two medical centers: taipei veterans general hospital and kaohsiung e-da hospital, taiwan. subjects were recruited by advertisement among medical employees, research laboratory staff working at both hospitals, and their relatives. all subjects gave informed consent before commencement of the study. the protocol was approved by the institutional review boards of the taipei veterans general hospital (taipei, taiwan) and e-da hospital (kaohsiung, taiwan). each subject was given an interview using a standard questionnaire to carefully review the history of medical disease, psychiatric illness, and medication use. subjects included in the study did not have a personal history of medical conditions (e.g., malignant tumors, heart failure, or diabetes mellitus), pregnancy, psychiatric illnesses or substance abuse/dependence. none of the subjects was taking any medication. the collected demographic data included age, sex, body mass index, and smoking. of note, most volunteers were hospital colleagues, and the rate of smoking was low (n = , . %). of these subjects, were successfully contacted for ambulatory electrocardiogram (ecg) monitoring. holter recordings (myecg e - portable recorder, microstar inc., taipei, taiwan) were used to obtain two hours of ecg signals. the e - device continuously recorded three channels of ecg signals at a sampling rate of hz. all ecg monitoring took place in the daytime, and participants were asked to avoid smoking or drinking alcoholic beverages and to stay in a resting state while being monitored. valid dna samples were obtained in subjects by drawing blood or by buccal swabs. the final study sample therefore consisted of healthy adult subjects ( males and females, aged . . years, range - years). among the present study sample, subjects have been included elsewhere in a previous report on the altered sympathovagal balance associated with val met polymorphisms of the bdnf gene [ ] . each subject was genotyped for three polymorphisms (rs , rs and rs ), and genomic dna was isolated using the puregene dna purification system (gentra systems, minneapolis, mn, usa). the genotypes of rs were determined using polymerase chain reaction and restriction fragment length polymorphism analysis. briefly, primers and probes were designed with spectrodesigner software (sequenom, san diego, ca, usa). pcr was then performed, and unincorporated double-stranded nucleotide triphosphate bases (dntps) were dephosphorylated with shrimp alkaline phosphatase (hoffman-laroche, basel, switzerland) followed by primer extension. the purified primer extension reaction product was spotted on to a -element silicon chip (spectrochip, sequenom) and analyzed in a bruker biflex iii maldi-tof spectro-reader mass spectrometer (sequenom). the resulting spectra were then processed with spectrotyper (sequenom). all samples were genotyped for eight unrelated snps for dna quality examination. the samples were diluted onto -well plates, and only the plates on which each of the eight unrelated snps had a successful genotyping rate greater than % were used for further study. all experiments were performed by investigators who were blind to phenotype. failure in genotyping for rs polymorphism was noted in cases. the ecg signals were automatically processed and analyzed by open-source hrv algorithms [ ] . the standard hrv analysis has been well reviewed [ ] . briefly, time domain measures of hrv include the mean heart rate and standard deviation of the normal interbeat intervals (sdnn), the root mean square successive difference between adjacent normal interbeat intervals (rmssd), and the percentage of adjacent intervals that varied by greater than ms (pnn ) [ ] . the sdnn assesses the overall variability of interbeat intervals. the rmssd and pnn measure the short-term variation of interbeat intervals, which is mainly modulated by parasympathetic innervation [ ] . standard spectral hrv measures [ ] include high-frequency power (hf; . - . hz), low-frequency power (lf; . - . hz), and very low-frequency power (vlf; . - . hz). lf power is suggested to be modulated by both sympathetic and parasympathetic activities, whereas hf power is mainly modulated by parasympathetic activity [ , ] . the lf/hf ratio is considered a measure of the shift of sympathovagal balance toward sympathetic activity [ , ] . the physiological mechanism underlying vlf power is disputed but has been suggested to be mediated partly by the renin-angiotensin-aldosterone system or parasympathetic modulation [ , , ] . in addition, we incorporated two nonlinear hrv indices: detrended fluctuation analysis (dfa) [ , ] and multiscale entropy (mse) [ ] . dfa quantifies the presence of long-range (fractal) correlations whereas mse measures the entropy over multiple time scales inherent in physiologic signals and is therefore a complexity measure. both methods are available at physionet (http://physionet.org), a research resource for complex physiologic signals [ ] . in the dfa method, the root-mean-square fluctuation of integrated and detrended time series is measured at different observation windows and plotted against the size of the observation window on a log-log scale. the scaling exponent a is then derived from the slope of line fitting to the obtained log-log plot. the short-term exponent a ( to heartbeats) and the longterm scaling exponents a (. heartbeats) were also calculated [ , , ] . low-exponent values represent reduced fractal propertiy of heart rate dynamics and have been implicated in the risk of fatal cardiac arrhythmia, increased mortality, or poor prognosis in cardiovascular diseases [ , , , ] . mse has been proposed as a biologically meaningful complexity measure by quantifying the entropy over multiple time scales inherent in physiologic signals. the procedure and calculation of the mse is summerized as following three steps: ) construction of coarse-grained time series, ) quantification of the sample entropy of each coarse-grained time series, and ) summation of the sample entropy values over a range of scales. in the present study, sample entropy was calculated using a pattern length (m) of and a similarity factor (r) of . . the sum of sample entropy over all scale factors from to was computed to represent the overall mse measure. in addition, the sum of sample entropy over scale factors from - and - was calculated to represent short-term and long-term mse measures, respectively [ ] . several methods of symbolic dynamic analysis of hrv have been proposed previously [ , , , , ] . the ibs method was developed to effectively categorize symbolic sequences according to their information content. the method has been fully described and validated [ ] , with applications to heart rate time series, literary texts, and genetic sequences [ , , ] . an interbeat interval time series (or heart rate time series) is mapped to a symbolic sequence, according to a mapping rule that accelerated heart rate in consecutive heartbeats is designated as and a deceleration of heart rate is designated as ( figure a ). this way of mapping captures the essential dynamics of the autonomic nervous system's control of heart rate and is less sensitive to noisy fluctuations in interbeat interval time series commonly caused by ectopic heartbeats [ ] . a binary, symbolic ''word'' is then defined as a n-tuple sequence derived from n+ consecutive interbeat intervals. we determined the frequencies of each pattern of n-tuple sequences by applying a sliding window (moving one interbeat interval/step) across the entire interbeat interval time series and then ranked each n-tuple sequence according to its frequency in descending order. to compare the similarity between symbolic sequences, we plotted the rank number of each n-tuple sequence in the first sequence against that of the second sequence ( figure b ). if two sequences are similar in their rank order of n-tuples, the scattered points will be located near the diagonal line (e.g., comparison between healthy subjects) [ ] . therefore, the average deviation of these scattered points away from the diagonal line is a measure of the dissimilarity index between these two sequences. we defined the distance (d n ) using n-tuples between two sequences, s and s , as here r (w k ) and r (w k ) represent the rank of a specific n-tuple, w k , in sequences s and s , respectively. n = n is the number of different n-tuple sequences (or patterns). the absolute difference of the mapping procedure for a n-tuple binary symbolic sequence (here n = for illustrative purposes) from part of an interbeat interval time series. (b) rank-order comparison of two interbeat interval time series from the two healthy subjects, using tuple binary symbolic mapping. in this case, frequencies of = tuple symbolic patterns were determined and ranked accordingly. for each -tuple symbolic sequence (black dot), its rank in subject a is plotted against its rank in subject b. the dashed diagonal line indicates the case where the rank order of -tuple symbolic sequence for both subjects is identical. doi: . /journal.pone. .g ranks, r (w k ){r (w k ) j j , is weighted by summing shannon's entropy h for w k in sequences s and s [ ] . shannon's entropy measures the information richness of each n-tuple in both sequences. thus, the more frequently used n-tuples contribute more to measuring similarity among symbolic sequences. of note, the ibs is an empirical index which does not necessarily obey the triangular inequality criterion of a distance measure. therefore, the triangular inequality test is required before generating a cluster [ , ] . the ibs algorithm was available at physionet (http:// www.physionet.org/physiotools/ibs/). the applications of the ibs method in the present study were two-fold: ) the pairwise distance of heart rate dynamics among individuals was calculated by an average of ibs distance using ntuple symbolic sequences (n = - ) and was used in subsequent hierarchical cluster analysis. ) to quantify the nonlinear aspect of heart rate time series, a non-randomness index was defined to measure the symbolic patterns of heart rate dynamics away from complete randomness (i.e., absence of ordered control of the autonomic nervous system) [ ] . the non-randomness index is independent of conventional entropic measures (i.e., sample entropy or approximate entropy) and has been evaluated in other studies [ , , , ] . the calculation of the non-randomness index is based on estimating the average n-tuple distance between raw interbeat interval time series and its randomly shuffled surrogates using the ibs method, where the number of surrogates was in the present study. the averaged non-randomness index derived from each n-tuple non-randomness index (n = - ) was then used in this study for comparisons among b-ar genotypes. we calculated allele and genotype frequencies and performed hardy-weinberg equilibrium tests for each b-ar genotype. the spectral hrv indices were log-transformed to produce normalized distributions. to control for the effects of non-genetic factors, differences in hrv variables were compared for individual genotypes using a general linear model (glm) followed by the bonferroni post hoc test for corrections of multiple between-group comparisons., using age, gender, and body mass index as covariates. effect sizes were calculated with cohen d, defined as the difference between the means of two groups, divided by the pooled standard deviation of these groups. partial correlation analysis was applied, controlling for age and bmi, to determine the associations between standard hrv indices and nonrandomness index derived from the ibs method. a p value of less than . (two-tailed) was required for all statistical comparisons. a complete-linkage, hierarchical clustering tree was estimated using the generalized association plot (gap) (http://gap.stat. sinica.edu.tw/). gap is implemented here as an unsupervised clustering algorithm to visually categorize all subjects based on the dissimilarity matrix, which is derived from calculating the pairwise dissimilarity of heart rate dynamics among subjects using the ibs method. cluster-specific association was analyzed by the chisquare test to assess the frequency of b-ar polymorphisms and by the glm model to test the differences in standard hrv indices among resulting clusters. to validate cluster-specific associations, we tested the differences in hrv and b-ar genotypes in estimated clusters based on first-and second-half ecg data. demographic and clinical data for subjects with each b-ar genotype are presented in table s and s . the subgroups in three genotypes did not differ in age, gender ratio, smoking status, or body mass index. there was no detectable deviation from hardy-weinberg equilibrium in b -ar ser gly genotype (x = . , p = . ), b -ar arg gly genotype (x = . , p = . ) or b -ar gln glu genotype (x = . , p = . ). tables , , summarize the association of b-ar genotypes with hrv indices determined by a glm model using age, gender, and bmi as covariates. there was no significant effect of interaction on hrv indices detected between b-ar genotypes and demographic variables. b -ar ser gly genotype was associated with mean heart rate and sdnn with borderline significance (table ; p = . and p = . , respectively). no statistical association was found between b -ar gln glu genotype and hrv indices ( table ) . for the b -ar arg gly genotype (table ) , a significant codominant association was found with lf% (f = . , p = . ) and the non-randomness index derived from the ibs method (f = . , p = . ). multiple comparisons showed that lf% was significantly lower in homozygous arg carriers compared to subjects with the arg /gly genotype (p = . ), but not to homozygous gly carriers (p = . ). likewise, a significantly lower non-randomness index was found in homozygous carriers of the arg allele (p = . ) compared to subjects heterozygous for arg /gly (p = . ), and a borderline significance was seen in the comparison to homozygous gly carriers (p = . ). there was no significant difference in hrv indices between heterozygotes arg /gly and homozygous gly carriers, correlations between non-randomness index and standard heart rate variability to ascertain the relationship of non-randomness index with autonomic function, we estimated the partial correlation between non-randomness index and standard hrv variables, controlling for age and bmi (table s ) . a weak but significant positive correlation of the non-randomness index with the lf/hf ratio was found (r = . , p = . ), whereas negative correlations were found with rmssd (r = . , p, . ) and pnn (r = . , p, . ). association of heart rate dynamic clusters with b-ar gene polymorphisms next, we investigated the b-ar genotype distributions in an unsupervised cluster tree based on the dissimilarity (distance) measure of heart rate dynamics. we first estimated the pairwise distance between interbeat interval time series of all subjects using the ibs method. no violation of the triangular inequality was observed. we then applied a hierarchical clustering algorithm to cluster these subjects based on the obtained distance matrix. two major clusters were identified in the resulting dendrogram ( figure ). there was no significant difference in age, gender, or other demographic variables between the two clusters (data not shown). table shows b-ar genotypes and standard hrv characteristics according to two major clusters. a significant deviation in the distribution of homozygous b -ar arg carriers was detected between the two clusters (p = . ), whereas the other genotypes showed no deviation in genotype distribution. in terms of standard hrv characteristics, cluster (with a higher rate of homozygous b -ar arg carriers) showed significantly reduced lf power (p, . ) and significantly increased hf power (p = . ). the clusters did not differ in other hrv indices. we verified the genotype distribution with clusters based on first-and second-half ecg data, and the results were consistent (table s ). the data presented in this study demonstrate a significant association of a common b -ar polymorphism, arg gly, with the non-randomness index, a nonlinear hrv measure derived from the ibs method. moreover, we illustrate that a bottom-up approach using the ibs method was able to measure the dissimilarity of heart rate dynamics among individuals, and we show that the resulting clusters were associated with b -ar arg gly genotype, indicating an impact of this b -ar polymorphism on acceleration/deceleration patterns of heart rate oscillations. our study offers several advantages for studying genetic associations with physiological parameters and might be generalizable to other genotype-phenotype studies based on different types of time series (e.g., brain electroactivity or time series derived from functional magnetic resonance imaging). first, our method enables us to cluster heart rate dynamics by an ibs method based on their acceleration/deceleration patterns of heartbeat sequence. the ibs method was not exclusively developed for the analysis of heartbeat sequence but also for other generic datasets consisting of repetitive elements [ , ] . with an appropriate mapping rule that is meaningful to a target dataset, the ibs method can be applied to other types of phenotypic data derived from the time series [ ] . second, we employed a visually aided hierarchical analysis by a software tool called a generalized association map, which enables a bottom-up approach to unsupervisedly identify heart rate clusters to study genotype-phenotype associations. this bottom-up approach may help reduce the false-positivity commonly seen in conventional association studies. b -ar is the predominant b-ar subtype in the heart. compared to activation of b -ar, stimulation in b -ar can result in more figure . unsupervised, hierarchical cluster tree of subjects according to the pairwise dissimilarity matrix among heart rate dynamics using an information-based similarity index method. dissimilarity matrix data were visualized and clustered by a generalized association plot algorithm [ ] . the bars on the left indicate the b-adrenergic receptor (b-ar) gene polymorphisms rs (green: homozygous ser carriers; blue: gly allele carriers; red: unknown genotype), rs (green: homozygous arg carriers; blue: gly allele carriers) and rs (green: homozygous gln carriers; blue: glu allele carriers). doi: . /journal.pone. .g significantly increased frequency and contractility of heart. although substantial efforts have been made to link b -ar polymorphisms with cardiovascular function, studies have been mixed in this regard. for example, b -ar ser gly polymorphism was found to be associated with increased blood pressure and heart rate [ , ] but data are inconsistent in our findings and other reports [ , ] . because we studied only one polymorphism in b -ar, further research is needed to identify other possible b -ar polymorphisms associated with hrv. this study found that b -ar arg gly genotype was associated with clusters based on acceleration/deceleration patterns of heart rate. although b -ar is expressed in the heart at lower concentrations than is the b -ar subtype, it also expresses on the presynaptic sympathetic nerve terminals and expresses abundantly in vascular and bronchial smooth muscle. therefore, changes in b -ar function may not only alter sympathetic activity [ ] , but also respiration and vascular responses [ , ] . it can be reasonable to speculate that these factors have influences in hrv. of note, the association between b -ar arg gly polymorphism and heart rate fluctuation patterns may simply reflect a functional genetic component nearby due to linkage disequilibrium and warrants future genetic research targeting on this region. the non-randomness index was developed initially as a nonlinear hrv index [ ] and its connection with other traditional hrv indices needs to be explored. though the nonrandomness index was weakly associated with hrv indices (rsquare, . ; table s ), it did correlate negatively with hf power and positively with the lf/hf ratio, suggesting that the non-randomness index is a marker related to sympathovagal balance. our findings of lower non-randomness index and lf% seen in homozygous b -ar arg carriers are in line with prior studies showing that the b -ar arg polymorphism is associated with lower sympathetic activity, manifested as lower blood pressure [ , ] , lower plasma norepinephrine [ , ] , and enhanced agonist-mediated desensitization in the vasculature [ ] . strength of this work include a larger sample size, matched gender distribution, and long recording of ecg signals using a holter recorder, compared to prior genetic association study of hrv. several limitations influence the interpretation of the findings presented in this study. first, we are unable to repeat findings of spectral hrv indices seen in a smaller male sample [ ] . this may be due to different gender distributions [ ] and means of ecg recording. twenty-four hour holter recording could validate and reinforce the association of b-ar genetic polymorphism with hrv seen in this study. second, we were unable to evaluate the correlation of the non-randomness index with other commonly used sympathetic indicators, such as blood pressure or plasma catecholamine levels. third, our findings are based on a healthy population and may not be generalizable to medically ill patients, such as those with cardiovascular diseases. the cross-sectional nature of our study also limited us examining the age modification of the effect of b-ar receptor polymorphism on autonomic function. in conclusion, this study shows that a bottom-up, categorization approach combining the ibs method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with b -ar gene polymorphisms. further research should aim to identify the physiological mechanisms underlying these findings. fractal dynamics in physiology: alterations with disease and aging heritability of heart rate variability: the framingham heart study heritability of ambulatory heart rate variability ethnic differences and heritability of heart rate variability in african-and european american youth genome-wide association study of electrocardiographic and heart rate variability traits: the framingham heart study inheritance of heart rate variability: the kibbutzim family study pleiotropy of creactive protein gene polymorphisms with c-reactive protein levels and heart rate variability in healthy male twins angiotensin-converting enzyme and angiotensinogen gene polymorphisms and heart rate variability in twins heart rate variability is associated with polymorphic variation in the choline transporter gene association of ucp and ucp polymorphisms with heart rate variability in japanese men association of beta-adrenoceptor polymorphisms with cardiac autonomic modulation in japanese males bdnf val met polymorphism alters sympathovagal balance in healthy subjects reduced physiological complexity in robust elderly adults with the apoe epsilon allele a polymorphism in the beta adrenergic receptor is associated with resting heart rate adrenergic receptor polymorphisms associated with resting heart rate: the hypergen study statistical physics approach to categorize biologic signals: from heart rate dynamics to dna sequences linguistic analysis of the human heartbeat using frequency and rank order statistics physiobank, physiotoolkit, and physionet: components of a new research resource for complex physiologic signals task force of the european society of cardiology and the north american society of pacing and electrophysiology: heart rate variability: standards of measurement, physiological interpretation and clinical use the pnnx files: re-examining a widely used heart rate variability measure relationship of heart rate variability to parasympathetic effect respiratory sinus arrhythmia: noninvasive measure of parasympathetic cardiac control assessment of autonomic function in humans by heart rate spectral analysis power spectrum analysis of heart rate variability: a tool to explore neural regulatory mechanisms mechanisms underlying very-low-frequency rr-interval oscillations in humans power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control quantification of scaling exponents and crossover phenomena in nonstationary heartbeat time series long-range correlations in nucleotide sequences multiscale entropy analysis of complex physiologic time series fractal analysis of heart rate dynamics as a predictor of mortality in patients with depressed left ventricular function after acute myocardial infarction. trace investigators. trandolapril cardiac evaluation dynamic analysis of heart rate may predict subsequent ventricular tachycardia after myocardial infarction predicting survival in heart failure case and control subjects by use of fully automated methods for deriving nonlinear and conventional indices of heart rate dynamics fractal analysis of heart rate variability and mortality after an acute myocardial infarction novel method to quantify loss of heart rate variability in pediatric multiple organ failure fractal correlation properties of r-r interval dynamics and mortality in patients with depressed left ventricular function after an acute myocardial infarction the prognostic value of non-linear analysis of heart rate variability in patients with congestive heart failure-a pilot study of multiscale entropy symbolic dynamics of heart rate variability: a probe to investigate cardiac autonomic modulation entropy, entropy rate, and pattern classification as tools to typify complexity in short heart period variability series quantitative analysis of heart rate variability short-term forecasting of life-threatening cardiac arrhythmias based on symbolic dynamics and finite-time growth rates nonlinear measures of heart period variability: decreased measures of symbolic dynamics in patients with panic disorder information categorization approach to literary authorship disputes genomic classification using an information-based similarity index: application to the sars coronavirus a mathematical theory of communication linear and non-linear indices of heart rate variability in chronic heart failure: mutual interrelationships and prognostic value nonrandomness index applied for heart rate variability in surgical intensive care units using frequency and rank order statistics nonlinear indices of heart rate variability in chronic heart failure patients: redundancy and comparative clinical value polymorphism in the beta( )-adrenergic receptor gene and hypertension effects of beta -adrenoceptor genetic polymorphisms on resting hemodynamics in patients undergoing diagnostic testing for ischemia overexpression of human beta -adrenergic receptors increases gain of excitationcontraction coupling in mouse ventricular myocytes beta-adrenoceptor polymorphisms and heart failure effect of adrb polymorphisms on response to longacting beta -agonist therapy: a pharmacogenetic analysis of two randomised studies arg gly polymorphism of the beta -adrenergic receptor is associated with differences in cardiovascular function at rest and during exercise in humans beta -adrenoceptor polymorphisms relate to insulin resistance and sympathetic overactivity as early markers of metabolic disease in nonobese, normotensive individuals beta -and beta -adrenergic receptor polymorphisms are related to the onset of weight gain and blood pressure elevation over years the effect of common polymorphisms of the beta -adrenergic receptor on agonist-mediated vascular desensitization influence of age, gender, body mass index, and functional capacity on heart rate variability in a cohort of subjects without heart disease methods for simultaneously identifying coherent local clusters with smooth global patterns in gene expression profiles key: cord- - s h j authors: ratnamohan, vigneswary m.; zeng, frank; donovan, linda; macintyre, chandini r.; kok, jen; dwyer, dominic e. title: phylogenetic analysis of human rhinoviruses collected over four successive years in sydney, australia date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: s h j background: human rhinoviruses (hrv) cause a wide spectrum of disease, ranging from a mild influenza‐like illness (ili) to severe respiratory infection. molecular epidemiological data are limited for hrv circulating in the southern hemisphere. objectives: to identify the species and genotypes of hrv from clinical samples collected in sydney, australia, from to . methods: combined nose and throat swabs or nasopharyngeal aspirates collected from individuals with ili were tested for hrv using real‐time reverse‐transcriptase polymerase chain reaction (rt‐pcr). sequencing data of ′utr and vp /vp coding regions on rt‐pcr‐positive specimens were analysed. results: human rhinoviruses were detected by real‐time pcr in . % ( / ) of samples tested. phylogenetic analysis of ′utr and vp /vp on hrv‐positive samples was concordant in the grouping of hrv a and b species but not hrv c species. eighty per cent ( / ) of sequences that grouped as hrv c in the vp /vp tree clustered as hrv a, alongside some previously described c strains as subspecies c/a. discordant branching was seen within hrv a group: two sequences clustering as a in the vp /vp tree branched within the c/a subspecies in the ′utr tree, and one sequence showed identity to different hrv a strains in the two genes. the prevalence of hrv c and c/a species was greater in paediatric compared to adult patients ( . % vs . %, p = . ). conclusion: human rhinoviruses are a common cause of respiratory infections, and hrv c is present in the southern hemisphere. sequencing of multiple hrv regions may be necessary to determine exact phylogenetic relationships. human rhinoviruses (hrv) are a diverse virus group, currently known to contain serotypes. along with human enteroviruses (hev), hrv belong to the picornaviridae family, although they are phylo genetically unrelated to hev despite similarities in genome organization and structure. human rhinoviruses are generally associated with the common cold and mild upper respiratory infections, but can also cause severe respiratory infections in immunocompromised hosts (including lung and hematopoietic stem cell transplant recipients) or patients with chronic pulmonary diseases. [ ] [ ] [ ] [ ] unlike respiratory syncytial virus (rsv) and influenza viruses, hrv can cause respiratory illness throughout the year, but peak incidence occurs in early autumn and spring in temperate climates. early molecular analyses of the hrv capsid protein coding regions clustered different serotypes into two distinct species, hrv a and hrv b. in , a new hrv genetic variant (subsequently designated hrv c) was identified in patients with severe pneumonia from the united states of america (usa), germany, hong kong, australia and china. [ ] [ ] [ ] [ ] [ ] severe respiratory disease and asthma exacerbations in children were observed. , hrv c has been further divided into two subspecies, hrv cc and hrv ca. hrv c strains are difficult to grow in cell lines known to support the growth of other rhinoviruses, although two hrv c isolates have been propagated in nasal epithelial cell cultures. there are limited molecular epidemiological data on hrv circulating in the southern hemisphere, including australia. this study aimed to identify the species and genotypes of hrv from clinical samples collected in sydney, australia, over four consecutive years by analysing the nucleotide homology in the ′utr, vp and part of the vp capsid protein coding regions. primers used to amplify all picornaviruses, the hrv-specific probes ′utr and vp -vp gene sequencing primers are listed in table . the rt-pcr probe sequences were designed to include most hrv sequences available in genbank ® in , and sequences generated from the present study. rna was prepared directly from combined nts or npa using roche high pure rna kits (roche, mannheim, germany). the cdna was reverse-transcribed from μl of specimen rna using units of superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa) and μl used for real-time pcr as described previously. amplification was performed in glass capillaries on human rhinoviruses rt-pcr-positive samples were then tested using utr primers ev and ev to amplify a -bp product. where rna was available, samples were amplified with primers rhi a and evp r, generating a -bp fragment that included part of ′utr, all of vp and part of vp regions. cs - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a •cs - -a a branches with hrv a- a ms - -a a a a ms - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a ms - -a a a a ms - -a a a a cs - -a a a a ms - -a a a a cs - -a a a a cs - -a a a a ms - -a a a a cs - -a a a a cs - - b a a a cs - - b a a a cs - - b a a a cs - - b a a a cs - - b a a a ms - -a a a a cs - -a a a a ms - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a ms - -a a a a ms - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a cs - -a a a a (continues) using blast ® , nearly all the sequences showed > % similarity to one or more hrv sequences with partial cds available in genbank ® . these were approximately - bp and did not cover the full sequence from ′utr to vp of our study samples, and were not included in the construction of the ml tree. as the data size was large, it was not possible to include each one of the hrv prototype sequences in the tree construction. figure shows the ml tree for the ′utr for all sequences, and figs and show the ml trees, respectively, of vp /vp and ′utr/vp /vp regions of the samples. in group c with vp /vp analysis also clustered in group c in the ′utr along with reference strain c-eu ; of the hrv c samples grouped as hrv a, along with reference strains c-x -ef , c-eu , c-dq , c-jq , c-ef , c-ef and c-gq . four clades that grouped as c species in the vp /vp analysis segregated into the a genogroup in the ′utr analysis (figs and ) . discordant branching was seen in the following and indicated by a circle figure ). samples with asterisk (*) denote strains that grouped as c in the vp /vp region, but grouped with hrv a species. samples with only ′utr sequences are denoted by ^. discordant branches in the vp /vp and ′utr sequences are indicated by • sample ms - and hrv a (ef ) clustered in a clade more related to c-dq (subspecies ca). the two samples showed between % and % id to c-dq strain and samples in that cluster and similar identity to a and its cluster in the utr tree, but in the vp /vp tree the two samples showed % identity to c-dq and lower to other c strains and % and % identity to a and b, respectively. maximum-likelihood tree of the ′utr/vp /vp region, showing relationships of clinical strains, newly described strains and prototype strains of hrv (constructed as described for fig. ). samples with asterisk (*) denote strains that grouped as c in the vp /vp region, but grouped with hrv a species in ′utr analysis. discordant branches in the vp /vp and ′utr sequences are indicated by • the sample sequences segregated into three phylogenetically distinct species: ( . %) hrv a, ( . %) hrv c and one ( . %) hrv b. several clades were represented within hrv a and c (fig. ) . within hrv a, identity at > % was seen with known hrv reference strains as shown in table the grouping of samples as c and ca in the ′utr/vp /vp analysis was very similar to that seen in the ′utr analysis. the discordant branching pattern seen with samples ms - and hrv a (ef ), ms - and ms - and cs - (indicated with •) in the ′utr ml tree was not seen; it was in agreement with that seen in the vp /vp ml tree. in the distribution of the different hrv subtypes is shown in table . both hrv a and c or c/a variant were detected in higher numbers than hrv b. human rhinoviruses c or c/a variant was detected more in our study, the sequences grouped into three phylogenetically distinct species: a ( . %), c/ca ( . %) and b ( . %). however, there was discordance between proposed phylogeny groups when sequences from the ′utr and vp /vp coding regions were analysed. sixteen of the samples that clustered as hrv c in the vp / vp ml tree segregated as a in the ′utr analysis, as did some of the early, well-characterized hrv c strains from new york (eu and dq ), , san francisco (ef and ef ), hong kong special administrative region (ef ) and china (gq ). twelve of sequences with only utr sequences also segregated along with the above-mentioned reference c strains, branching in two major subgroups within hrv a. these have been labelled in this study as c/a, clustering with c strains, but grouping as a in the ′utr region: these formed % of the clinical sequences. huang et al. reported similar clustering of field strains in their study and designated the above c strains (eu , dq , ef , ef , gq and ef ) as ca, a subspecies of hrv c that clustered differently to hrv a, hrv b and hrv c in the utr. they further showed that hrv ca subspecies were formed from interspecies recombination in the ′utr region. similar inconsistent clustering of field strains as compared to vp /vp was also reported in the d polymerase-coding region as well as ′utr region. three discordant branching events were seen in our analyses. ms - (identity to a ) in one branch and ms - along with hrv a in another branch segregated as a in the vp /vp analysis, but localized within the ca subspecies in the ′utr analysis. sample cs - clustered with hrv a with % relatedness in the utr region but differed in the vp /vp region. these three samples may represent recombinants as reported by palmenberg et al. and kim et al. , there is no designated region within the hrv genome that is , the clustering of hrv c as c and c/a when ′utr region was included in the analysis (figs and ) in our study may suggest that they are true c species that showed recombination with a in the ′utr. in this study, the ′utr primers and the primers chosen to amplify the entire vp and partial vp region produced amplicons with overlapping sequences that resulted in an approximately bp continuous sequence. we used a single set of primers for each of the two pcr assays but did not use cloning, which has been used in other studies. were collected from patients with respiratory illness who were sick enough to warrant testing or in many cases hospitalization, but our data are insufficient to attribute clinical severity to any of the hrv it is possible that hrv c or the c/a variants may cause exacerbation of respiratory infections in infants that require presentation to ed compared to hrv a or b, and this may contribute to the higher percentage of hrv c or c/a infection in the paediatric population. one of the early reports of hrv c-qpm variant severity was from australian samples collected in from children with lower respiratory infection. other reports have observed the association between hrv c variants and asthmatic wheeze and severe lower respiratory infections. , , , , xiang et al. reported that the clinical manifestations of hrv a and c are similar, and co-infection with rsv and hrv in infants increases the severity of infection. both hrv a and c are more virulent than hrv b in infants and hrv virulence is greater in winter, although peak infection rates occur in spring and fall. in conclusion, the present study shows that sequencing of one region alone is insufficient for determining the lineage of the hrv variants. the presence of many diverse strains has become apparent, and it is likely that more will emerge. genotypic assignment and identification of hrv types will facilitate monitoring of emerging novel variants, and investigations into type-associated differences in disease epidemiology, transmission and outcomes. samples used in this study were not collected for this study per se, but as part of a study assessing the use of face mask in controlling respiratory virus transmission in households following approval by the local institutional review board or for laboratory diagnosis of patients with an influenza-like illness. the present study does not involve the reporting of patient data, and no patient intervention occurred with the obtained results. virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses a collaborative report: rhinoviruses-extension of the numbering system from to viruses and bacteria in the etiology of the common cold rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients human rhinoviruses human rhinovirus c in adult hematopoietic stem cell transplant recipients with respiratory illness genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus human rhinovirus species and season of infection determine illness severity a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) human rhinovirus group c infection in children with lower respiratory tract infection assay for ′ noncoding region analysis of all human rhinovirus prototype strains evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c face mask use and control of respiratory virus transmission in households detection of human picornaviruses by multiplex reverse transcription-pcr and liquid hybridization pandemic clinical case definitions are non-specific: multiple respiratory viruses circulating in the early phases of the influenza pandemic in new south wales enterovirus is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses detection and differentiation of picornaviruses in clinical samples following genomic amplification a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity new respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses human rhinovirus c: age, season, and lower respiratory illness over the past decades proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types analysis of complete genome sequences of human rhinovirus molecular characterization of human rhinovirus field strains isolated during surveillance of enteroviruses sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution identification of recombinant human rhinovirus a and c in circulating strains from upper and lower respiratory infections clinical and molecular features of human rhinovirus c analysis of genetic diversity and sites of recombination in human rhinovirus species c human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons molecular characterization and distinguishing features of a novel human rhinovirus (hrv) c, hrvc-qce, detected in children with fever, cough and wheeze during a novel group of rhinoviruses is associated with asthma hospitalizations we thank ms. gordana nedeljkovic for compiling patient information from the laboratory information system network. funding: none.competing intesrests: none declared. vmr participated in the study design, analysed the results and drafted the manuscript. fz and ld performed nucleic acid testing. crm was the chief investigator of the study on the use of face masks to reduce household transmission of respiratory viruses. jk had input in the preparation and editing of the manuscript. ded participated in the design of the study and was involved in manuscript editing. all authors have read and accepted the manuscript. additional supporting information may be found online in the supporting information tab for this article. key: cord- -zgapjjjw authors: faux, cassandra e.; arden, katherine e.; lambert, stephen b.; nissen, michael d.; nolan, terry m.; chang, anne b.; sloots, theo p.; mackay, ian m. title: usefulness of published pcr primers in detecting human rhinovirus infection date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: zgapjjjw we conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (hrv)–specific pcr primer pairs, varying the oligonucleotides and annealing temperature. none of the pairs could detect all hrvs in panels of genotyped clinical specimens; > pcr is required for accurate description of hrv epidemiology. h uman rhinoviruses (hrvs) cause more asthma exacerbations than any other known factor, in addition to causing most colds and infl uenza-like illnesses. the prevalence of hrv in published reports varies considerably. a novel hrv clade identifi ed in , now known as hrv species c (hrv-c) ( ), can be identifi ed only by pcr. since , seasonality and clinical outcomes and numerous different primer pairs have been used to identify hrv; how well these methods perform on new hrv types is uncertain. given the likely variation in the preparation of rna, the quality and formulations of commercial reverse transcription (rt)-pcr enzymes and reaction mix components and changes in thermal cyclers since , not surprisingly many, perhaps most, of these assays are not being used in the manner they were originally described. for example, the fi rst hrv-specifi c primers reported ( ) have subsequently been used with different rna preparation methods, amounts of reverse transcriptase, cdna priming strategies, dntp concentrations, annealing temperatures (t m s), and cycling conditions ( , ) . we conducted a preliminary comparison of the relative sensitivity of a cross-section of published hrv-specifi c pcr primer pairs (most of which were fi rst published before hrv-c was reported), independent of most variables described above, by testing a panel of clinical specimen nucleic acid extracts from combined nose and throat swabs from preschool children with colds and infl uenza-like illnesses in melbourne, australia. the study was approved by the royal children's hospital human research ethics committee. the panel included representatives of the hrv species (figure) , human enteroviruses (hevs), and extracts negative for picornaviruses. the hrvs had been previously detected by using a nested primer pair (online appendix table, www.cdc.gov/eid/content/ / / -appt.htm) ( ). we used different hrv primer pairs and also retested specimens by using the original primer pair with our standard reagents and equipment ( ) . we applied the published t m when possible. the original descriptions of primer pairs and (online appendix table) lacked t m information, and after in-house calculations, we used t m s of °c and °c, respectively. we also deliberately standardized the reagents (onestep rt-pcr kit, qia-gen, doncaster, victoria, australia) and thermal cyclers used (veriti, applied biosystems, foster city, ca, usa) for conventional pcr and the rotorgene real-time cycler (qiagen). because primer pair had a published history of detecting types from all hrv species, we chose it to genotype hrv-positive samples by sequencing the amplifi ed products. other pairs were used if pair was unsuccessful. we found that no primer pair detected the same hrvs and hevs typed when the original pair ( ) or pair (online appendix table) was used. five primer pairs, including real-time pcr (rtpcr) pair , did not amplify the hevs, a positive feature for hrv-specifi c studies. only primer pairs amplifi ed anything from a specimen that was positive for both hrv and hev, a problem for accurate estimation of the frequency of co-detections. the original primer pair screen detected untypeable picornaviruses, which were not detected by any other pair or by repeat testing using the same pair. only the second-round amplicon of the nested sets of nested primer pairs ( , , and ) was considered because the second round increased the total number of positive specimens over the fi rst round. the longest amplicon, produced by primer pair , was also a valuable genotyping target, but it detected only of the original hrv-positive specimens in this population. we next selected frequently published primer pairs ( , , , and ) to examine picornavirus-positive specimens ( hrvs, hevs, and untypeable picornaviruses) from nonhospitalized children with acute asthma exacerbation ( ) . as before, primer pair detected the greatest num- most notably, primer pair performed better than it had in the previous population, detecting only fewer hrv than primer pair and more hrvs than pair . no speciesspecifi c bias was apparent, but generally, a specimen with a lower rna concentration, as indicated by the cycle threshold from primer pair , was less likely to be detected or typed by using other primer pairs. primer pairs and did not detect the hevs (hev- ). we noted in both populations that primer pair sometimes amplifi ed a region of human genomic dna from chromosome (gq ), for which amplicon size was indistinguishable from that expected due to hrv. it was not possible to use the precise conditions reported for the compared assays; was published > decades ago and used phenol chloroform extraction. some of the original enzyme formulations or reagents are no longer available, and production processes have changed in the interim. thermal cyclers have also changed. there was no consensus on enzymes and reaction mixes used. in addi-tion, the previously published primers were used in assays divided between those using -step rt-pcr and those using a separate rt cdna synthesis step. a review of studies that detected hrvs with adequately described conditions during - found that fewer used a single-tube rt-pcr approach than a -step system. we conducted singletube rt-pcr to maintain the benefi ts of the so-called closed amplifi cation system of rtpcr. thus, we chose to use a single common set of reagents as the fairest way to compare the primer pairs examined in this study. we believe the nature of this relative comparison best refl ects performance for the likely end users: clinical microbiology laboratories or researchers. we compared primers rather than assay function using clinical material instead of cultured virus, plasmid or synthetic rna standards, or screening contemporary or archived extracts, which are sometimes of low viral load. when picornavirus epidemiology is the primary research focus, we recommend using > primer pairs to maximize the detection of hrvs. under our conditions, pairs - returned the highest number of positive results, and the rtp-crs behaved similarly but with reduced sensitivity. the rtpcr that used pair did not amplify known hevs. many possible reasons could cause discrepant virus testing results between different sites, including changes to specimen integrity resulting from transport and variable amplifi cation resulting from low viral loads. the effects of viral load can be seen in this study: specimens in population that were positive with multiple (> separate pairs) primer pairs had a mean cycle threshold of . (combining results from both rtpcrs), whereas those with < positive results had means of . cycles. most ( / ) specimens with < positive primer pairs were negative by rtpcr. amplifi cation variability can also be attributed to the substantial nucleotide sequence diversity between hrvs and the different temporal and clinical characteristics of the specimen populations we used. population diversity is a feature of hrv studies in the literature. our selection of published primer pairs includes those from studies that have informed our current understanding of hrv epidemiology. finding such a high degree of variability in performance was thus noteworthy. ineffi cient hrv detection by pcr may be a serious problem for research studies. comparison of data between different hrv studies is confounded as are data from studies seeking to determine the effects of other respiratory viruses. the prevalence, seasonality, transmission, and clinical effects of hrv types and species require reexamination with tools that have been comparatively validated to ensure their sensitivity. figure. distribution of human rhinovirus (hrv) and human enterovirus (hev) sequences used for primer pair studies. the hrv and hev genotypes from the testing panel (indicated by fi lled circles) were aligned with the central nt of the ′ untranslated region (utr) region of all complete hrv genomes and poliovirus- . hrv-ca and hrv-cc refer to hrv-cs with ′ utr sequences that have phylogenetic origins from either hrv-as or hrv-cs, respectively. the tree was constructed by neighbor joining of maximum composite likelihood distance implemented in mega (www.megasoftware.net). frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections amplifi cation of rhinovirus specifi c nucleic acids from clinical samples using the polymerase chain reaction rhinoviruses replicate effectively at lower airway temperatures picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens newly identifi ed respiratory viruses in children with non-hospitalised asthma exacerbation polymerase chain reaction amplifi cation of rhinovirus nucleic acids from clinical material early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses new respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus identifi cation of enteroviruses in clinical specimens by competitive pcr followed by genetic typing using sequence analysis simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays real-time rt-pcr detection of respiratory viral infections in four triplex reactions key: cord- -t hoox e authors: dearden, c. j.; al‐nakib, w. title: direct detection of rhinoviruses by an enzyme‐linked immunosorbent assay date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: t hoox e this paper describes the first enzyme‐linked immunosorbent assay for the detection of rhinovirus antigens in clinical specimens (nasal washings), either directly or following overnight cell culture amplification. the assay takes approximately hours to perform and utilizes the same rabbit antirhinovirus hyperimmune serum as both the capture and detecting antibody. the latter has been biotin‐labelled and is detected via a streptavidin ‐galactosidase preformed complex. this new assay has been found to be very sensitive, detecting human rhinovirus (hrv)‐el and hrv‐ at titres as low as ( . ) tcid( ) μl(− ) and < ( ) tcid( ) μl(− ), respectively. furthermore, when different human rhinovirus serotypes were tested in both the hrv‐el and hrv‐ elisa systems a total of ( %) were found to be cross‐reactive. of clinical specimens tested by virus isolation, cell‐culture‐amplified (cca) elisa, and direct elisa, were positive by isolation, by cca‐elisa, and by direct elisa. the overall correlation of the cca and direct elisa techniques with virus isolation was found to be . % and . %, respectively. the present study demonstrates that the elisa system developed is a sensitive technique for the diagnosis of rhinovirus infections. rhinoviruses are the major causative agent of the common cold and could be responsible for at least half of all acute respiratory infections in humans [mogabgab, ; higgins et al, . most rhinovirus infections are trivial and self-limiting in that only a minor upper respiratory tract illness of some - days duration occurs. however, in a proportion of cases, particularly in immunocompromised individuals and children with a history of bronchitis and allergy, infection can be more serious [craighead et al, . in addition, there is some evidence to suggest that rhinovirus infections are associated with exacerbations of bronchitis and asthma [gregg, . furthermore, krilov et a have recently detected rhinoviruses in out of ( %) specimens from infants with lower respiratory tract infection [krilov et al, . rhinoviruses are therefore appropriate targets for treatment with antiviral chemotherapeutic agents. rapid diagnosis of rhinovirus infection will be necessary so that antiviral treatment can be initiated. however, at present, virus isolation and identification, in an appropriate cell-culture system, may take up to weeks to complete. we have therefore developed an enzyme-linked immunosorbent assay (elisa) so that this time interval can be reduced to approximately hours. in this test the capture and the detecting antibody are both prepared from the same rabbit hyperimmune antirhinovirus serum. the latter, however, is labelled with biotin and is then detected using a streptavidin- -galactosidase preformed complex. this system was proven to be both sensitive and specific. in this paper we describe an assay which can be used to detect rhinoviral antigens both directly in nasal washings and following overnight cell culture amplification of the same specimen. the results obtained were compared with those yielded by conventional virus isolation procedures. the rhinovirus used to establish the elisa procedure was the untyped human rhinovirus el. although a member of the rhinovirus genus according to its physical and biochemical characteristics, hrv-el has not yet been assigned a serotype number in the collaborative scheme [kapikian et al, ; kapikian et al, . hrv-el was grown to high titre by infecting confluent monolayers of ohio human epithelial carcinoma (hela) cells, in -oz medical flats, overlaid with -ml of hela maintenance media (bme containing % foetal calf serum, . % tryptose phosphate broth, . % nah co,, mm mgcl,, and antibiotics). after the cytopathic effect had developed, the virus was released from the cells by three cycles of freezing and thawing. the tissue culture fluid containing the virus was then clarified by low-speed centrifugation ( , rpm for minutes) and stored at - ooc. control antigen was prepared in the same way except that the cells were not infected. all the other rhinoviruses used in this study were passaged once from laboratory stocks. ohio hela cells, grown to confluence in -cm plastic tissue culture flasks (sterilin), were infected with virus and overlaid with -ml hela maintenance media. the tissue culture fluids were then harvested as for hrv-el and stored at - ooc. the titre of all the rhinovirus stocks were estimated by titration in ohio hela cells. the high titre hrv-el, prepared as an antigen, was further purified by fluorocarbon extraction. one volume of arklone (ici) was shaken, at room temperature, with two volumes of antigen; and the mixture was then centrifuged at , rpm for minutes. the aqueous layer was harvested and centrifuged at , rpm for hours. the virus pellet was then resuspended in a small volume at . m nacl/o. m tris buffer (ph . ) and mixed with an equal volume of freund's incomplete adjuvant. rabbits were immunized with the purified antigen by inoculating . ml of the emulsion intramuscularly in each hind leg followed by ml intravenously weeks later. the animals were bled week later, and the hyperimmune serum was found to have a neutralisation titre of : , against tcid,, of the homologous virus. a hyperimmune serum to hrv- was prepared following the same schedule and was found to have a neutralisation titre of : against tcid,, of the homologous virus. a small aliquot of the hyperimmune serum was dialyzed overnight at °c against three changes of . m nahc (ph . ) (b.d.h.). in ml of dimethylsulphoxide (sigma) . mg of biotin (n-hydroxy-succinimidobiotin) (sigma) was dissolved. two hundred microlitres of the biotin preparation was added to each millilitre of the hyperimmune sera, and the mixture was allowed to react for hours, mixing slowly on a laboratory stirrer, at room temperature. the mixture was then dialyzed overnight at °c against three changes of . m phosphate-buffered saline (ph . ) (difco) [gary et al, . eighteen volunteers inoculated intranasally with hrv-el or saline, who took part in a separate trial at the common cold unit [zerial et al, . provided samples for this study. nasal washings were collected on days after virus challenge by instilling millilitres of hanks' balanced salt solution into each nostril. the expelled fluid was mixed with an equal volume of nutrient broth and stored at - °c. virus in nasal washings was isolated in ohio hela cells. the streptavidin wash (amersham) was supplied as a mg ml-' solution in mm phosphate buffer (ph . ) containing . % w/v sodium azide and was diluted to a working concentration ( / ) with phosphate buffered saline (ph . ) (difco) for the test. the streptavidin preformed complex was supplied as streptavidin-biotinylated p-galactosidase complex (amersham) and was diluted to a working concentration ( / ) with phosphate buffered saline (ph . ) (difco) for the test. ortho nitrophenyl-p-d-galactosidase (onpc) (sigma), a water-soluble yellow product that absorbs light at nm, was used as the basis of the substrate and prepared for the test as follows: . mg ml-i onpg, mm mgcl, (b.d.h.), and . m -mercaptoethanol (sigma) in phosphate buffered saline (ph . ) (difco) [craven et al, . all assays were performed in rigid, nonsterile, u-bottomed, -well polystyrene plates (cibco/nunc). wells were coated overnight at oc with pl of rabbit hyperimmune antirhinovirus-el serum diluted / , with . m carbonate-bicarbonate buffer (ph . ) (elisa coating buffer, don whitley scientific ltd). plates were emptied and pl of % v/v bovine serum albumin (bsa) (sigma) in pbs was added for hours at °c in order to block any nonspecific binding. the hrv-el antigen, which had a titre of approximately lo tcid,, pl-l, was diluted in log,, steps in pbs-tween + . % v/v bsa + % v/v control antigen. the control antigen (uninfected tissue culture fluid) was also diluted in the same way. plates were washed three times with pbs-tween ( p per well) using a -outlet minimicrowash (skatron) and p of hrv-el or control antigen was added to a set of wells and plates were then incubated overnight at °c. after washing as before, pl of the streptavidin wash, diluted / with pbs, was added to the wells in order to block any nonspecific binding to endogenous biotin in the specimens. the plates were incubated at room temperature for minutes, followed by five washes with p per well of pbs-tween. one hundred microlitres of biotinylated antiserum, diluted to its optimal working dilution with pbs-tween + . % v/v bsa + % v/v control antigen, was added and the plates were incubated at oc for hours. after three washes as before with pbs-tween, pl of streptavidin / -galactosidase preformed complex, diluted / with pbs-tween + . % v/v bsa, was added to the plates, which were again incubated at °c for hours. plates were then washed five times with pbs-tween, pi per well, and pl of substrate (prepared approximately minutes prior to use) was added to all the test wells plus a row of wells, not used in the test, as a blank. plates were left covered at oc and read hourly at nm using an automatic plate reader (titertek, multiscan, flow laboratories) until the colour intensity exceeded its maximum. as each dilution of hrv-el or control antigen was added to a duplicate set of wells, the mean optical density of each set was calculated. a positive result was recorded when the mean optical density of the test wells was . times the mean optical density of a similar dilution of control angiten. this "cutoff" value was set after experiments were performed in which a series of negative and positive specimens at various dilutions were assayed. mean optical densities recorded for the negative specimens never exceeded . times the mean optical density of the control antigen (undiluted), whereas the mean positive specimen optical densities were always > . times the mean control antigen optical density. a similar assay for hrv- was also developed using the same procedure as described for hrv-el. the optimal dilutions of the reagents used in the elisa systems were determined by checkerboard titrations. experiments to determine optimal incubation times and temperatures were performed once the reagent concentrations had been standardized. cross-reactivity experiments were performed with a total of different human rhinoviruses and four other control viruses (influenza a/eng/ / , coronavirus , echovirus , and coxsackie a ) plus control antigen (uninfected tissue culture fluid) as controls. the titre of all the viruses was adjusted to approximately lo .' tcid ml-' by diluting with pbs-tween + . % v/v bsa + % v/v control antigen, whereas the control antigen was used undiluted in the test. the antigens were then tested in both the hrv-el and hrv- elisa systems according to the described protocol. the "cutoff" value was calculated as stated in the elisa procedure. therefore, a positive result was again recorded when the mean optical density (od) of a duplicate set of sample wells was . times the mean optical density calculated for the control antigen (undiluted). nasal washings, taken on consecutive days, from the volunteers challenged with hrv-el or saline were tested for the presence of hrv-el antigen in the hrv-el elisa system. initially a cell culture amplification procedure was employed in which p of each nasal washing was inoculated, in duplicate, into monolayers of ohio hela cells grown in microtitre plates (nunc microwell plate f with lid). after minutes of adsorption at oc, the nasal washings were removed and pl hela maintenance media was added to each well. the plates were incubated at oc overnight. after one cycle of freezing and thawing pl from each test well was removed and placed in the corresponding well of a precoated elisa plate. the plates were then incubated overnight at "c, and the test continued according to the elisa protocol. secondly, a direct elisa system was developed in which the nasal washings or control antigen (uninfected tissue culture fluid) were added directly to each of a set of duplicate elisa plate wells coated with either pre-or postchallenge rabbit anti hrv-el hyperimmune serum. plates were then incubated overnight at °c and the test continued as described above. test results were calculated by subtracting the mean optical density of a specimen tested in duplicate pre-serum-coated wells from the mean optical density of the same specimen tested in duplicate post-serum-coated wells. results for the control uninfected tissue culture fluid were also calculated in this way. a nasal washing was considered positive when the test result was . times that of the control uninfected tissue culture fluid. this calculation was found to be the most appropriate method of controlling the considerable variation in background optical densities obtained with different nasal wash specimens. the "cutoff" value was set after a series of positive and negative specimens had been assayed using the direct elisa as described in the elisa procedure. a % bsa block and a streptavidin wash stage were incorporated in the test in order to block unoccupied sites on the surface of the elisa plate and endogenous biotin in clinical specimens, respectively. experiments were performed in which each stage of the test was omitted in turn and the results compared with that obtained from a complete test. figure shows that the % bsa block and streptavidin wash were necessary stages in the elisa test in order to keep background levels to a minimum. with the optimal test conditions established, experiments were performed in order to determine the limits of detection of the assay for both hrv-el and hrv- antigen detection. the optical density, recorded at nm, decreased with increasing dilutions of the hrv-el and hrv- stocks and remained approximately the same for all dilutions of control antigen. at a titre of lo'.' tcidso pl-' and <lo' tcid,, pl-' hrv-el and hrv- , respectively, the mean optical density was approximately equal to . times that recorded for control antigen. therefore the limits of detection of the assay were concluded to be tcid,, pl-' and <lo' tcid,, pl-' for hrv-el and hrv- , respectively. studies on the cross-reactivity between the various rhinovirus serotypes are summarised in table . briefly, of the different human rhinoviruses tested, ( . %) showed strong cross-reactivity, ( . %) showed a lesser degree of cross-reactivity, ( . %) were weakly cross-reactive, whilst ( . %) showed no reaction at all with hrv-el. all four control viruses (echovirus type , coronavirus e, coxsackie a and influenza a/eng/ / ) were not cross-reactive. control antigen also showed no reaction. in the hrv- elisa system, ( . %) of the rhinoviruses tested (including hrv- ) showed strong cross-reactivity. twelve ( . %) showed a lesser degree of cross-reactivity, ( . %) were weakly cross-reactive, whilst ( . %) showed no reaction. of the controls, coronavirus e, coxsackie a , influenza a/eng/ / , and control antigen did not cross-react with hrv- . however, echovirus type reacted strongly in the test, thus suggesting a degree of cross-reactivity with hrv- . these results were reproduced several times. figure shows the results obtained with nasal washings, collected from three volunteers on consecutive days following hkv-el or saline challenge, tested in both the cell-culture-amplified (cca)-elisa and direct elisa systems. the figure also illustrates the good correlation obtained between cca-elisa, direct elisa, and virus isolation in detecting positive or negative specimens from these three volunteers. nasal washings collected or days after inoculation with hrv-el or saline from volunteers who participated in a trial were investigated by all three methods strong" moderateb weak' negatived , , , el , , , , , , , , . , la, ib, , , , , , , , , , , , , , , , , , , , , , , , , echo i, , , . , coronavirus e, influenza , , , , , , , , , , , , , , , , , la, ib, , , , , , , , , . , , , , , , , , , , , , , , , , , , , , , , , , , . x the od of control antigen. "weak = od of test sample . - x the od of control antigen. dnegative = od of test sample i l . x the od of control antigen. (table ). samples from of ( . %) volunteers gave concordant results by all three procedures for both days. in two cases (ad and ag) specimens were positive by isolation but negative by cca-elisa. however, overall there was . % correlation between the cca-elisa and virus isolation. the direct elisa failed to detect viral antigen in specimens that were positive by isolation and specimens that were positive by both isolation and cca-elisa. however, viral antigen was detected in specimens that were negative by isolation and cca-elisa. hence, the direct elisa showed . % agreement with either virus isolation or it is noteworthy that all the control nasal washings collected from the four volunteers who were challenged with saline instead of hrv-el (n.o., a.c., j. ram, j.r.) were negative by all three procedures (table ii) , thus indicating the high specificity of the elisa techniques. cca-elisa. in this paper we describe the first elisa system for rhinovirus detection. the assay was found to be simple, rapid, and capable of detecting a large number of human rhinovirus serotypes. it is a reliable method for the detection of hrv-el antigen in clinical specimens, and results have suggested that the limits of detection were as low as lo -' tcid,, loopl-' and <lo tcid,, pl-l for hrv-el and hrv- antigen, respectively. the assay offers three distinct advantages over virus isolation in cell culture. firstly, the test is rapid, requiring only hours to complete once antibody coated plates are available whereas virus isolation and serological identification may take as long as weeks. secondly, the elisa, as demonstrated in this study, is capable of detecting noninfectious as well as infectious virus (some volunteers showed r -fold rises in antibody titre, were elisa positive yet did not excrete virus); hence, the need to preserve infectivity of virus in the clinical specimen may not be essential. thirdly, as tests are read spectrophotometrically evaluation of results is objective rather than subjective. a biotin-avidin elisa utilising the enzyme ,f -galactosidase was developed in this study because of the following advantages over conventional elisa. the high affinity of avidin for biotin (affinity constant = mol-') [green, ensures that the biotinavidin complexes are not easily dissociated by the washing steps in the test and hence this may result in enhanced sensitivity [guesdon et al, . furthermore; one molecule of avidin is capable of binding four molecules of biotin and biotin can be covalently linked to antibody without affecting the antigen binding capacity [guesden et al, . it is more specific as the enzyme p-galactosidase prepared from escherichia coli and used in this assay has characteristics different from those found in human tissues under the conditions of the assay. therefore by employing a -galactosidase-based substrate (onpg), high background readings-as seen with alkaline phosphatase or horseradish peroxidase-were avoided. the data presented in this paper demonstrate that a large number of human rhinovirus serotypes are cross-reactive in both the hrv-el and hrv- elisa systems. this would suggest that an antirhinovirus serum is capable of binding not only its homologous virus but also a number of heterologous viruses. however, the degree of cross-reactivity depends on how close the serological relationship is between the test virus and that against which the hyperimmune serum was prepared. nevertheless, our data clearly suggest that it may be possible to prepare a hyperimmune serum capable of detecting many rhinoviruses by including a number of rhinovirus serotypes in the initial inoculum. indeed, in this study we demonstrated that by using the two elisas (for hrv-el and hrv- ) we were able to detect a large number of the human rhinoviruses investigated (od x od of control antigen). alternatively, it may be possible to develop hyperimmune sera or monoclonal antibodies with c-type reactivity capable of detecting an even larger number of rhinovirus serotypes. although we would like to emphasise that our data are preliminary, both the direct and cca-elisas gave a good correlation with virus isolation when used to detect rhinovirus antigens in nasal washings (obtained from volunteers challenged with either hrv-el or saline). however, the cca-elisa showed better correlation ( . %) with virus isolation than the direct assay ( . %). this was not totally unexpected as both virus isolation and the cca-elisa depend on the presence of viable virus rather than antigen. the inclusion of an amplification stage in the elisa system may be necessary when virus is present at a low concentration (<lo tcid,, ml-i), as is often found with nasal washings obtained from adults. the addition of an amplification stage in the assay does not, in our opinion, increase the work or time load of the test, as whilst the elisa plates are precoated overnight with the capture antibody nasal washings are inoculated into monolayer cell cultures. recent studies with cytomegalovirus (cmv) and influenza virus have suggested that cell culture amplification followed by immunofluorescent staining was necessary for the detection of virus present at low concentration in urine [griffiths et al, ; alpert et al, or throat swabs [espy et al, , respectively. since rhinoviruses are also excreted at low concentrations, particularly by adults, and as nasal washings (although the optimal method of collecting a rhinovirus specimen for virus isolation) contribute further to the dilution of the excreted virus, we feel that the cca-elisa may be the method of choice for the detection of rhinoviruses when present at low concentrations. it is noteworthy that the time required to investigate serial specimens from volunteers using conventional virus isolation and neutralisation techniques is some to weeks. however, when our elisas are employed this time interval is reduced to hours. in addition, by reacting the same clinical specimen with both a pre-immunisation and a hyperimmune rabbit serum as capture antibodies, as in the direct method, the elisa can offer an in-built confirmation stage. it is therefore our aim to further modify the elisas described in this paper in order to improve sensitivity and decrease the time required to obtain results to within a few hours [same day diagnosis]. these developments will be essential if antiviral chemotherapy becomes available and a rapid diagnosis is therefore required in order to initiate treatment in individuals who may be at risk of developing more serious lower respiratory tract complications following rhinovirus infection. rapid detection of human cytomegalovirus in the urine of rapid detection of influenza virus by shell vial assay detection of norwalk virus antibodies with a ): . biotin-avidin immunoassay provocation of airflow limitation by viral infection: implication for treatment rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci the use of avidin-biotin interaction in immunoenzymatic techniques viruses associated with acute respiratory infections in royal air force personnel rhinoviruses: a numbering system a collaborative report: rhinoviruses-extension of a numbering system the association of rhinoviruses with lower respiratory tract disease in hospitalized patients acute respiratory illness in university ( - ), military and industrial ( - ) populations studies on r.p., a new antirhinovirus compound, in cell cultures and in volunteers. antimicrobial agents and chemotherapy we are very grateful to dr. d.a.j. tyrrell for helpful discussions and advice. we thank dr. r.j. phillpotts for preparing the rabbit hyperimmune sera, and mrs. n. bailey and mrs. l. treagust for performing virus isolation and neutralisation tests. key: cord- - cyz o c authors: barclay, wendy s.; callow, kathleen a.; sergeant, marianne; ai‐nakib, widad title: evaluation of an enzyme‐linked immunosorbent assay that measures rhinovirus‐specific antibodies in human sera and nasal secretions date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: cyz o c rhinovirus‐specific antibodies have traditionally been detected by their ability to neutralise the homologous rhinovirus serotype in tissue culture. recently, however, we have described an enzyme‐linked immunosorbent assay that detects rhinovirus‐specific antibodies in sera and nasal secretions [barclay and al‐nakib, ]. here we describe an evaluation of the elisa in a study involving adult volunteers inoculated intranasally with human rhinovirus type (hrv‐ ). pre‐and post‐inoculation serum samples and pre‐inoculation nasal washings were tested for the presence of hrv‐ ‐specific antibodies by elisa. such antibodies were associated with protection against infection when present locally in nasal secretions, but when also present in the serum they were associated with protection against both infection and the development of illness. the antibody concentrations showed strong correlation with each other and with that of antibodies detected by the neutralisation test. following hrv‐ infection, rises in hrv‐ ‐specific iga in sera detected by elisa occurred more frequently than rises in neutralizing antibody. these results suggest that the elisa is a sensitive and reliable indicator of recent infection, as well as a predictor of homologous immune status. rhinoviruses are the major causative agents of the common cold [couch, . earlier studies suggested that, following infection, rhinovirus serotype-specific antibodies appear in both the serum and nasal secretions and that the presence of such antibodies confers long-term protection against re-infection by the homologous rhinovirus serotype [doggett, . these antibodies have traditionally been detected by neutralisation tests [hamparian, . recently, however, we have established a sensitive enzyme-linked immunosorbent assay that detects rhinovirus-specific antibodies in sera and unconcentrated nasal washings. this new assay is simple, rapid, and can differentiate between classes of rhinovirus-specific immunoglobulins. in a preliminary study involving volunteers inoculated intranasally with human rhinovirus el (hrv-el), we showed that the presence of circulating rhinovirus-specific iga, rather than igg, protected volunteers from infection [barclay and al-nakib, . in the present study we describe a more extensive analysis of the anti-human rhinovirus type (anti-hrv- ) immunoglobulin responses in both sera and nasal washings measured by elisa. thus volunteers were inoculated intranasally with hrv- , and the course of infection was monitored both clinically and virologically . our data confirm the importance of iga anti-rhinovirus immunoglobulins in protection against re-infection and/or illness; we provide evidence that a rise in specific serum iga following infection is a good indicator of a recent infection. samples were obtained from volunteers taking part in a separate study to test the therapeutic effect of zinc gluconate lozenges on colds caused by hrv- [al-nakib et al., . they were housed in isolation according to standard procedures at the common cold unit [beare and reed, . serum samples were collected before the trial and weeks after rhinovirus inoculation. these were stored at - °c. nasal washings for antibody measurement were collected before the trial by instilling ml of phosphate-buffered saline (pbs) into each nostril. the volunteer then forcibly expelled the liquid into a pot containing glass beads. after vortexing for sec to break up mucus, the effluents were centrifuged at , rpm in a microcentrifuge, and the supernatants were stored at - °c. nasal washings for virus isolation were taken from to days after inoculation using standard methods [barclay and al-nalub, . these were added to an equal volume of nutrient broth and stored at - °c. viruses were isolated by inoculation of ohio hela cells. samples of . ml nasal washing were inoculated into confluent test tube cultures, these were rolled at °c. after - days they were examined for the presence of virus cytopathic effect (cpe). colds were assessed by a combination of tissue count and subjective and objective evaluation of severity of symptoms, that is, clinical score [beare and reed, . on the basis of virus isolation and these criteria, volunteers were divided into three groups for analysis as follows: ) colds group: those from whom virus was isolated and who had clinical colds; ) subclinically infected group: those from whom virus was isolated but who did not have colds; ) not infected group: those from whom no virus was isolated and who did not have colds. human rhinovirus type (hrv- ) was grown in ohio hela cells and crude antigen preparations were made as previously described for hrv-el [barclay and al-nakib, . control antigens was prepared in the same way from uninfected monolayers . fifty millilitres of crude hrv- antigen with a titre of approximately lo .' tcid /ml was concentrated by ultracentrifugation and resuspending the virus pellet in ml of pbs; . ml of this antigen was then emulsified in an equal volume of freund's complete adjuvant (fca) and used to inoculate one rabbit subcutaneously. six weeks later, the rabbit was inoculated intramuscularly with an emulsion of . ml hrv- and . ml of freund's incomplete adjuvant (fica). fourteen days later, the animal was bled out. the resulting hyperimmune serum had a neutralisation titre of : , against lo tcidso hrv- . this was stored at - °c. the elisa for hrv-zspecific immunoglobulins in sera and nasal secretions was an adaptation of that previously described for hrv-el-specific immunoglobulins [barclay and al-nakib, . that is, polystyrene round-bottomed micro elisa plates (nunc immunoplates u type ) were incubated overnight at °c with pl per well of a : ,ooo dilution of rabbit anti-hrv- hyperimmune serum in carbonate/bicarbonate buffer, ph . . wells were drained and washed three times in pbs containing . % tween (pbs-t). then pl of hrv- or control antigen (cag) diluted : in pbs-t containing % bovine serum albumin (bsa) was added to alternate rows of wells. plates were incubated for hr at "c, then drained and washed as before. one hundred microlitres of standard or test sample diluted in pbs-t containing % bsa and % cag was added in duplicate to both hrv- -and cag-coated wells. as standard, a pool of known hrv- -antibody-positive sera or nasal washings was titrated on each plate in one-half loglo dilution steps. samples were diluted to - . and respectively, for the assay of specific iga and igg in sera and for the detection of specific iga in nasal washings. plates were then incubated for hr at °c. after washing as before, p of either anti-human iga alkaline phosphatase conjugate (sigma) or anti-human igg alkaline phosphatase conjugate (icn) was added, diluted : ,ooo in pbs-t with % bsa, and incubated overnight at °c. plates were finally washed four times with pbs-t and p substrate (sigma p-nitrophenol phosphate tablets dissolved at mg/ml in % diethanolamine buffer) was added to each well. plates were incubated for hr and optical densities (od) at nm were read in a dynatech mr elisa reader using a reference filter of nm. mean ods from duplicate wells were corrected by subtracting the mean od of cag-coated wells. the serum standards were arbitrarily allotted . loglo units and the nasal washing standards, . loglo units. using corrected ods, from the titrations of these on each elisa plate a standard curve was calculated. corrected ods for each sample were then read off on this standard curve. thus each sample was awarded a number of loglo units, which related it to the known standards. antibody units for hrv- specific iga in nasal washings were further corrected by normalising for total iga in the sample. the elisa for total iga in nasal washings was performed as previously described by callow [ . this normalisation resulted in a more significant difference between the specific iga concentrations in nasal secretions for the three groups. to establish the size of a significant rise in hrv- -specific antibody following hrv- infection, paired sera from volunteers who had received an intranasal inoculation of saline (no virus), and who had been housed in isolation exactly as the other volunteers, were tested for hrv- antibodies. a significant rise in hrv- specific antibody was then set as the mean difference between these paired sera plus sd of that mean. thus a significant rise for elisa hrv- -specific iga in sera was a difference of . loglo units, and for igg serum-specific antibody this value was . log lo units. a rise in hrv- -neutralising antibody was considered significant if the difference was fourfold (i.e., . loglo steps) or greater. neutralising antibodies in sera were detected by microneutralisation tests against a challenge of tcidso homologous rhinovirus as previously described [barclay and al-nakib, . the statistical analyses were performed on an ibm xt computer, using the programme, statistical package for personal computers (spp, patrick royston, clinical research centre, northwick park hospital, harrow, england). for comparing the frequencies of antibody rises in the three volunteer groups, a x test was used, with yates' correction. for examining the significance of the differences in antibody concentration between the groups, a rank analysis of variance was used; for comparing antibody concentrations of different types, or the magnitude of antibody rises, spearman's rank correlation coefficient was calculated. of the volunteers who were intranasally inoculated with hrv- , ( . %) had colds, ( . %) had subclinical infections, and ( %) did not become infected. figure shows a) the mean hrv- -neutralising antibody, b) specific igg and c) iga in the serum, and d) specific iga in nasal secretions (normalized for total iga) in pre-inoculation samples from the three groups of volunteers. the presence of hrv- -specific antibody in the serum appears to confer protection against both infection and symptoms. thus volunteers who did not get infected had more hrv- -specific antibody in their sera (iga and igg antibodies) than those who had subclinical infections or those who were infected and developed colds. those who developed colds had the lowest amount of hrv-zspecific antibody in their sera. these differences in amounts of hrv-zspecific serum antibody between the three groups were highly significant for elisa-specific iga and neutralising antibody ( p < .ool) but were not significant for elisa-specific igg. volunteers volunteers were divided into these three groups according to their subsequent response to hrv- inoculation, as described in the text. who were not infected had significantly more hrv- -specific iga in pre-inoculation nasal washings ( ' < . ) than those who became infected whether or not they had colds. however, the differences between the concentrations of this antibody when the three groups were considered individually, or between the colds group and the subclinically infected group were not significant. therefore, although the presence of local specific antibodies appears to protect against infection, they do not appear to prevent illness caused by the infection. table i shows the number of volunteers in each of the three groups who had a significant rise in hrv- -specific serum antibody following hrv- intranasal inoculation. the frequencies of rises in neutralising antibody and elisa-serum-specific iga were significantly different in the three groups (p < . ), the proportion of rises being highest in the colds group, and least in the not infected group. the difference in frequency of rises in specific igg in the three groups was not significantly different. thirty-six of the volunteers showed rises in both elisa-serum-specific iga and neutralising antibody, but only two volunteers showed rises by neutralisation and not by elisa. in contrast, nine volunteers showed rises by elisa and not by neutralisation (table ) . thus the rises in elisa-serum-specific iga were slightly more frequent than for neutralising antibody ( of or . % vs. of or . %), suggesting that the former might be a more sensitive test for detecting such rises. the concentrations of the various hrv- -specific antibodies were tested for correlations with each other and with the size of antibody rises. substantial correlations were found between the concentrations of serum antibodies and between rises in these antibodies, as shown in figure a . generally the presence of, or a rise in, neutralising or elisa serum igg or iga antibody was significantly associated with the presence of, or rise in, the other rhinovirus-specific antibodies. there was also a positive correlation (p < . ) between the presence of hrv- -specific iga in nasal secretion and that in the serum. in addition, a negative correlation was found between the concentration of specific iga in nasal secretion and the size of the rise in specific iga in serum ( p < .ol) (fig. b ). thus volunteers with low amounts of pre-inoculation hrv- specific iga in nasal secretions were those most likely to show a significant rise in serum-specific iga. similarly, there was also a negative correlation between the concentration of specific iga in pre-inoculation sera and rises in rhinovirus-neutralising antibody and vice versa ( p < .ool). this suggests that a low specific iga or neutralising antibody concentration in pre-inoculation sera was associated with subsequent large rises in both neutralising antibody and specific iga following inoculation with hrv- . we have found a significant association between the presence of neutralising antibody and elisa-serum-specific iga and protection against re-infection and illness. thus, volunteers who had high levels of hrv-zspecific iga or neutralising antibody in their sera did not get infected or develop symptoms of colds when inoculated with hrv- . in contrast, those who had lower levels of specific iga or neutralising antibody had subclinical infections, and those who had little or no antibody became infected and developed colds. interestingly, the presence of hrv- specific iga in nasal secretions was only associated with protection against infection but not illness. our findings are in general agreement with those for another virus that causes common colds-coronavirus e [callow, . however, in that study, serum-specific igg appeared to be at least as effective in preventing infection and disease as serum-specific iga. furthermore, coronavirus-specific iga in nasal secretion, while significantly shortening the period of virus shedding, also appeared to reduce symptoms. recently, a similar study involving samples obtained from volunteers inoculated with another rhinovirus serotype (hrv- ) and tested by elisa for homologous antibodies, appears to confirm the association between elisa-serumspecific iga and protection against reinfection (callow and sergeant, unpublished data). the standard serological test for a recent rhinovirus infection is the detection of a rise in neutralising antibody. this study showed that rises in circulating rhinovirusspecific iga were at least as reliable for this purpose. in fact, the present elisa detected rises in iga in three subclinically infected and four uninfected volunteers, in whose sera the neutralisation test failed to detect such rises. these elisa antibody rises may have been "false," since the "uninfected" volunteers did not excrete virus. in a separate study in our laboratory, however, several nasal washings from these hrv- -inoculated volunteers from which no virus was isolated (i.e., the not infected group) contained hrv- antigen detectable by a biotidstreptavidin bridge elisa system. thus, although they were not excreting infectious virus that could be isolated in ohio hela cells, they were actively producing viral antigens, which suggests that viral replication may have been taking place. the size of rises in hrv- -specific iga in the serum after virus inoculation showed a significant negative correlation with the concentration of specific iga in the pre-inoculation nasal secretions and with the presence of serum neutralising antibody, indicating that volunteers who were most susceptible to infection and illness, in fact, showed the largest antibody response. the small number of serum-specific igg rises detected by the elisa and the weak correlation between the presence of pre-inoculation igg and protection against re-infection might be explained by the fact that, generally, the elisa detects large amounts of specific igg in all volunteer pre-inoculation sera so far tested. we suggested previously that this may be due to the use of crude tissue culture fluid harvests as antigens in the elisa that contain a mixture of bothjkll or native virus particles and empty capsids. the latter have been shown to cross-react with antisera against heterologous serotypes [lonberg-holm and yin, . thus, igg persisting from previous infections with other rhinovirus serotypes could cross-react in the elisa. we have recently obtained evidence that purification of the homotypic full virus particles on sucrose density gradients for use as antigens in the assay improves the specificity of the test (barclay, unpublished data) . this cross-reactivity does not appear to be a problem when an iga class-specific conjugate is used, presumably because iga antibody responses are of short duration and do not persist as long as those of the igg class. in conclusion, the elisa described in this study is an accurate predictor of the jmmune status of an individual to the homologous rhinovirus and can be a reliable diagnostic assay detecting recent rhinovirus infection by measuring significant rises in specific iga. thus it could be useful in evaluating the efficacy of any novel rhinovirus vaccine or antiviral compound in the future by measuring the incidence of infection following challenge with the homologous virus. we are now studying the possibility that this elisa system can be modified to detect rises to other rhinovirus serotypes. prophylaxis and treatment of rhinovirus colds with zinc gluconate lozenges an elisa for the detection of rhinovirus specific antibody in serum and nasal secretion chemoprophylaxis and virus infections of the respiratory tract effect of specific humorai immunity and some non-specific factors on resistance of volunteers to respiratory coronavirus infection the common cold: control? the journal of infectious diseases prevention of colds by vaccination against a rhinovirus: a report by the scientific committee on common cold vaccines diagnostic procedures for viral, rickettsia and chlamydia infections antigenic determinants of infective and inactivated human rhinovirus type we are very grateful to dr. d.a.j. tyrrell for helpful discussion and advice. we thank miss morag forsyth, miss caroline dearden, and mrs. patricia chadwick for performing virus isolations, and mrs. nicola bailey for collecting samples. miss wendy barciay is supported by an mrc studentship. miss marianne sergeant is supported by nih grant no. r a - . key: cord- - pgcfk b authors: sidoti, francesca; bergallo, massimiliano; terlizzi, maria elena; piasentin alessio, elsa; astegiano, sara; gasparini, giorgio; cavallo, rossana title: development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes date: - - journal: mol biotechnol doi: . /s - - - sha: doc_id: cord_uid: pgcfk b evidence demonstrating that human rhinovirus (hrv) disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of hrv infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of hrv infections. in this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. we described a simple method to accurately quantify rna target by computing the time to positivity (ttp) values for hrv rna. quantification capacity was assessed by plotting these ttp values against the starting number of target molecules. by using this simple method, we have significantly increased the diagnostic accuracy, precision, and trueness of real-time nasba assay. specificity of the method was verified in both in silico and experimental studies. moreover, for assessment of clinical reactivity of the assay, nasba has been validated on bronchoalveolar lavage (bal) specimens. our quantitative nasba assay was found to be very specific, accurate, and precise with high repeatability and reproducibility. human rhinoviruses (hrvs) are the most frequent cause of acute upper respiratory tract infections in humans and are usually responsible for - % of cases of common cold [ ] [ ] [ ] . however, they may also be associated with moresevere lower respiratory tract infections. rhinoviruses have been isolated from cases of cystic fibrosis, otitis media, sinusitis, asthma, exacerbations of chronic obstructive pulmonary disease (copd), and pneumonia, especially in children, in the elderly, and in immunocompromised patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . evidence demonstrating that hrv disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of hrv infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of hrv infections. two nucleic acid amplification techniques (naats) are actually available for the detection of hrv: reverse transcription-pcr (rt-pcr) [ , ] , and nucleic acid sequence-based amplification (nasba) [ , ] . nasba has proven to be highly sensitive, specific, and more rapid than rt-pcr technique [ , ] . currently, only qualitative nasba kits for the detection of hrv are commercially available (registered trademarks owned of biomérieux, marcy l'etoile, france), while there are no quantitative nasba kits. some quantitative molecular beacon real-time nas-ba assays have been described in the literature, mainly for the identification of human immunodeficiency virus (hiv), respiratory syncytial virus a and b, influenza a virus (h n ), trypanosoma brucei, aspergillus fumigatus, plasmodium species, and listeria monocytogenes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, these real-time nasba assays use mathematical models for the analysis of results that requires employing of specific complex software calibrated to each target [ ] . other quantitative nasba assays, instead, compute the ratio of the time to positivity (ttp) values for both the target rna and internal control by using standard curves with a correlation coefficient less than . (r \ . ), index of an insensitive assay [ , , ] . aim of this study was to develop the first quantitative real-time nucleic acid sequence-based amplification assay internally controlled using molecular beacon for selective and sensitive detection of hrv serotypes. validation and standardization were performed by evaluating diagnostic trueness, precision, and accuracy of real-time nasba assay. prototype human rhinovirus serotype (hrv- ) was obtained from the american type culture collection (atcc, manassas, virginia). rhinovirus serotype (atcc vr- ), originally isolated from a human clinical specimen, was extracted by using an automatic extractor nuclisens easymag platform (biomérieux, france), according to the manufacturer's recommendations. onehundred-microliters of hrv- were used for the extraction, rna was eluted in ll of nuclease-free water and stored at - °c. to evaluate the specificity of the hrv nasba assay, purified rna templates from hrv isolates, and selected respiratory viruses other than hrv were used for inclusivity and exclusivity testing (table ) . in vitro rna transcription viral cdna was generated, first by incubation of random primers ( ng/ll) and dntps ( mm) (invitrogen) with ll of hrv- rna for min at °c. subsequently, a mix containing buffer [ mm tris-hcl (ph . at °c), mm kcl, and mm dtt], mgcl ( mm), impromii rt ( u/ll), and recombinant rnasin Ò ribonuclease inhibitor ( u/ll) (promega) was added. the total volume ( ll) of the reaction mixture was incubated for min at °c, min at °c, and min at °c using fast thermal cycler (applied biosystems, monza, italy). crna production was carried out using t -ribomax large scale rna production systems (promega, usa) at °c for h. one-tenth of crna product was treated with rq rnase-free dnase (promega, usa) at °c for min followed by incubation with edta for min at °c. hrv- crna was purified using rnagent kit (promega, usa) following manufacturer's instructions. hrv- crna was quantified using quant-it dna br assay on qubit tm fluorometer (invitrogen, carlsbad, usa), and the number of molecules per microliter calculated from the molecular weight of hrv- amplicon ( , mw) and avogadro number ( . ). ten-fold dilutions of rna standards were generated in order to amplify from to copy per reaction, and frozen at - °c until use. for the production of internal control (ic) rna, we used the human u a housekeeping gene encoding the ''a'' protein present in the human u small nuclear ribonucleoprotein (snrnp) particle. to generate the ic rna, the u a molecule was extracted from a clinical specimen, precisely from a human bronchoalveolar lavage sample, and subjected to reverse transcription (rt) reaction by using random primers ( ng/ll) and impromii rt ( u/ll). ic cdna product was amplified by using adapted nasba primers containing the t rna polymerase promoter site. briefly, ll of ic cdna was added to ll of pcr solution containing flexi buffer , mm mgcl , unit gotaq Ò hot start polymerase (promega), lm of each dntp, and lm of each u a primer. after an initial denaturation step of min at °c, the first-round pcr amplification was carried out under the following conditions: °c for s, °c for s, °c for s for cycles, then one cycle at °c for min using the fast thermal cycler (applied biosystems). pcr product was transcribed in vitro using t -ribomax, as previously described (see ''in vitro transcription'' in the ''materials and methods'' section). ten-fold dilutions of ic rna standards were generated in order to amplify from to copy per reaction. primers and molecular beacon probes were obtained from literature [ , ] (table ) , synthesized by eurogentec (seraing, belgium), and diluted to a final concentration of lmol/l. moreover, to maximize the oligonucleotides stabilization, we added % dimethyl sulfoxide (dmso) to primers and beacons mixture. the hrv beacon was labeled with fam at its -end and quencher dabcyl at its -end, while the ic beacon contained a rox at its -end and a dabcyl quencher at the -end. the stability and predicted structure of the beacons were analyzed by using the european mfold server (http://frontend. bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form .cgi). real-time nasba reaction was performed on a nuclisens easyq analyzer (biomérieux) using the nuclisens easyq basic kit version (biomérieux, lyon, france) for the amplification according to the manufacturer's manual. to obtain the best amplification efficiency, conditions for the real-time nasba assay were optimized until the best primers, beacons, and kcl concentrations were determined. titrations of ic rna (between and copies) and hrv rna were performed to determine the optimal amount of internal control to generate the greatest dynamic range for the assay without interference with the detection of hrv rna (data not shown). as a result, each reaction was run with the addition of copies of the ic rna. briefly, a total volume of ll of reaction mixture containing mm kcl and . lm of the hrv-and ic-specific primers was incubated with . ll hrv rna and . ll ic rna in the presence of . lm of hrvand ic-molecular beacons at °c for min to denature secondary structure rna. the reaction was subsequently cooled to °c for min to anneal the primers before adding ll of enzyme mixture containing avian myeloblastosis virus retrotranscriptase, rnase h, and t rna polymerase. after a brief centrifugation and gentle mixing by tapping, the tubes were then incubated at °c for min. to estimate the dynamic range of the real-time nasba assay (range of concentrations over which the method performs in a linear manner with an acceptable level of trueness and precision), we used hrv standard dilutions from copies/ll to copy/ll. sensitivity of nasba assay was determined by the lowest standard dilution consistently detectable in replicate reactions at frequency of %, whereas the limit of detection by the lowest concentration of target quantified. nuclease-free water was included as the no-template control (ntc) to serve as a control for background fluorescence. the optimal real-time nasba assay conditions that allowed efficient amplification of the hrv target were established. sensitivity and limit of detection of nasba assay were assessed by repeated testing of serial logarithmic dilutions of the hrv rna standards ranging from to copy/ reaction. in particular, hrv nasba dynamic range was calculated from ttp value (time point at which emitted fluorescence exceeds the baseline emission) regressed against the standard curve by using an excel spreadsheet created ''ad hoc'' by us (table ) . results from linear regression show that hrv real-time nasba assay was able to quantify from to copies/reaction. the standard curve of hrv dilutions plotted versus nasba amplifications (expressed as tpp) is shown in fig. , whereas plots for the amplification of hrv standard dilutions (from to copies/reaction), and the optimal amount of ic rna ( copies/reaction), are shown in fig. . the consistency of replicates was measured by the correlation coefficient (r ), which indicates the linearity of tpp values plotted in the standard curve. the r index for hrv was . . sensitivity of real-time nasba assay was determined by the lowest standard dilution consistently detectable in replicate reactions at frequency of %. hrv sensitivity was copies/reaction, whereas the limit of detection for reliable quantification was copy/ reaction. based on the data available at the blast alignment software, primers were tested in silico for potential crossreactivity with respiratory viruses other than hrv, and demonstrated no significant homologies to any other sequences. moreover, hrv primer and probe set, tested on respiratory viruses, was able to detect only hrv isolates, thus being the inclusivity of % (table ). the assay's specificity was further demonstrated by its ability to exclude all respiratory viruses other than hrv listed in table . no positive results were demonstrated for the other respiratory viruses, indicating that this molecular assay is highly specific for hrv isolates, thus being the exclusivity of % (table ) . diagnostic trueness of hrv real-time nasba method, defined as the degree of agreement between the average value obtained from a large series of test results and an accepted reference value, was evaluated. to establish the level of trueness and concordance with the assigned value, data from replicate measures of each dilution that we performed ( , , , and ) were analyzed using a student's t test to compare the mean concentrations from each dilution with an accepted reference value. the mean concentrations from each dilution for the method are shown in table with the t test results, which indicate the significance of the differences between each experimental mean and the assigned value. analysis of the t statistics showed that the method had t-calc values lower than the t-tab value, demonstrating a significant trueness of hrv assay. precision of method was expressed as the coefficient of variation (cv) in the log values of the concentration. repeatability and intermediate reproducibility of hrv assay were evaluated over different concentrations ranging from to copies/reaction from replicate measures (n = ) of each reference viral quantification standard within a single run or in different run experiments performed by three different operators. the precision associated with each dilution measurement ( , , , and ) was assessed by calculation of the cv for each. the cvs within a single run (repeatability) ranged from . to . %; whereas, the cvs in different runs (intermediate reproducibility) ranged from . to . % (table ), indicating that the precision of the assay is satisfactory. diagnostic accuracy includes both trueness and precision. the measure of accuracy is usually expressed numerically in terms of bias (lack of agreement). accuracy shall be within ± % of the accepted reference value over the whole dynamic range, according to document iso [ ] . data for the percentage of inaccuracy hrv method are reported in table . the developed real-time nasba assay was able to detect hrv rna in / ( . %) bal specimens. the ttp values of these nasba-positive samples ranged between and min when plotted against the standard curve in fig. (data not shown) . all the results were validated by the addition of internal control rna to rule out inhibition of amplification. in all cases, amplification of the control rna was observed, thus, confirming that all the negative and positive results are valid. moreover, all negative control reactions were nasba negative, demonstrating the absence of amplicon contamination. viral respiratory tract infections have been recognized as a predominant cause of human disease. to improve clinical management of such patients, it is important to obtain an accurate diagnosis and to identify the causative agent early in infection to ensure appropriate treatment. in this study, we developed the first quantitative nasba assay for the detection of hrv serotypes. by combining nasba amplification with molecular beacon probes, this assay becomes a real-time analysis tool that offers faster results than conventional rt-pcr technique. since nasba amplification involves three separate enzymes each with their own kinetic parameters, variability in every measurement is inevitable [ ] . weusten et al. [ ] were the first to describe a mathematical model for rna amplification of both target and internal calibrator rna in a molecular beacon-based nasba reaction to normalize enzyme efficiency differences between reactions. however, the description of this model did not include all of the essential parameters needed to operate the model. consequently, analysis using this model requires software calibrated to each target and is commercially available for only a few specific targets. here, we describe an alternative method for normalizing nasba data by using a simple tpp calculation in the presence of an internal control that reduces the variability between replicates and increases the precision, trueness, and accuracy for predicting unknown concentrations of hrv rna. it has been shown that tube to tube variation within nasba can be normalized with the addition of an internal control in each reaction [ , ] . in particular, the optimal concentration for the internal calibrator should be determined as the concentration providing the greatest dynamic range of amplification of both the target and internal control rna. in our study, a fixed amount of copies of the internal calibrator rna was optimal for the assay reported here (data not shown). furthermore, the addition of an internal control is fundamental to identify false negative results because of reaction failure, and to monitor the effects of unknown sample factors that might interfere with the amplification kinetics. as concerns the importance of primers and kcl for nasba optimization, their role has not been emphasized to date. nasba is an isothermal nucleic acid amplification method able to specifically amplify target rna by using specific primers in a kcl background. initially, the concentration of the primers relative to the total concentration of amplifiable rna is very high, is not rate limiting, and relatively small amounts of primers are consumed in depletion of the initially present pool of rna copies (linear phase of nasba process). at some time point, obviously, the primers' concentrations do become rate limiting, and decline to practically zero. at this time point, the dna intermediate levels have reached their maximum and rna production proceeds at high speed. from now on, the only reaction that can proceed is t rna polymerase-mediated formation of rna from the dna intermediate templates. this time interval represents the second phase of nasba process characterized by an exponential kinetics. we observed that high concentrations of primers and kcl elongate the linear phase of nasba process by shorting the exponential amplification; whereas, low concentrations of primers and kcl promote the exponential phase. in particular, in this study we used relatively low concentrations of primers and kcl ( . lm and mm, respectively) to elongate the exponential phase of nasba process, and accordingly, to minimize the reaction-toreaction variation. by using this simple expedient, we have significantly increased our accuracy, precision, and trueness of prediction over the standard ttp calculations. in summary, we describe a simplified method of calculating unknown concentrations of target rna using an internal calibrator. this method allowed for greater precision, accuracy, and trueness for predicting hrv rna over the standard ttp analysis. in conclusion, we described the first real-time nasba assay capable of accurate quantification of hrv rna making it a valuable tool in the molecular diagnostics of hrv serotypes. respiratory virus infections and aeroallergens in acute bronchial asthma recurrent wheezy bronchitis and viral respiratory infections viruses and bacteria in the etiology of the common cold rhinovirus in acute otitis media improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients. clinical infectious diseases conventional respiratory viruses recovered from immunocompromised patients: clinical considerations polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction development of a rt real-time pcr for the detection and quantification of human rhinoviruses development and application of a new method for amplification and detection of human rhinovirus rna improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the noncoding regions of additional rhinovirus strains increased precision of microbial rna quantification using nasba with an internal control detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season real-time nucleic acid sequencebased amplification is more convenient than real-time pcr for quantification of plasmodium falciparum evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type detection and identification of human plasmodium species with real-time quantitative nucleic acid sequence-based amplification rapid and highly sensitive qualitative realtime assay for detection of respiratory syncytial virus a and b using nasba and molecular beacon technology a molecular beacon-based real time nasba assay for detection of listeria monocytogenes in food products: role of target mrna secondary structure on nasba design detection of trypanosoma brucei parasites in blood samples using real-time nucleic acid sequence-based amplification quantitative determination of plasmodium vivax gametocytes by real-time quantitative nucleic acid sequence-based amplification in clinical samples detection of novel swine origin influenza a virus (h n ) by realtime nucleic acid sequence-based amplification detection of aspergillus fumigatus in a rat model of invasive pulmonary aspergillosis by real-time nucleic acid sequence-based amplification principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons the role of respiratory viruses in cystic fibrosis human cytomegalovirus virions differentially incorporate viral and host cell rna during the assembly process microbiology of food and animal feeding stuffs. protocol for the validation of alternative method. iso/dis usefulness of quantitative nucleic acid sequence-based amplification for diagnosis of malaria in an academic hospital setting molecular-based methods for quantifying hiv viral load key: cord- -zs l s authors: leotte, jaqueline; trombetta, hygor; faggion, heloisa z.; almeida, bernardo m.; nogueira, meri b.; vidal, luine r.; raboni, sonia m. title: impact and seasonality of human rhinovirus infection in hospitalized patients for two consecutive years date: - - journal: jornal de pediatria doi: . /j.jped. . . sha: doc_id: cord_uid: zs l s abstract objectives to report epidemiological features, clinical characteristics, and outcomes of human rhinovirus (hrv) infections in comparison with other community acquired respiratory virus (crv) infections in patients hospitalized for two consecutive years. methods this was a cross-sectional study. clinical, epidemiological, and laboratory data of patients hospitalized with acute respiratory syndrome in a tertiary care hospital from to were reviewed. results hrv was the most common crv observed ( %, / ) and was present in the majority of viral co-detections ( %, / ), mainly in association with human enterovirus ( %). most hrv-infected patients were younger than years ( %). overall, patients infected with hrv had a lower frequency of severe acute respiratory infection than those infected with other crvs ( % and %, respectively, p = . ), but had more comorbidities ( % and %, respectively; p = . ). however, in the adjusted analysis this association was not significant. the mortality rate within the hrv group was %. detection of hrv was more prevalent during autumn and winter, with a moderately negative correlation between viral infection frequency and temperature (r =− . , p < . ) but no correlation with rainfall (r =− . , p = . ). conclusion hrv is usually detected in hospitalized children with respiratory infections and is often present in viral co-detections. comorbidities are closely associated with hrv infections. these infections show seasonal variation, with predominance during colder seasons. human rhinoviruses (hrvs) belong to picornaviridae family, genus enterovirus, and are divided in three species (hrv-a, hrv-b, and hrv-c) with about serotypes within these species. , the development of highly-sensitive molecular techniques for characterization of the hrv genome has recently allowed recognition of the hrv-c species. there is already evidence that this new species may be more virulent and more strongly associated with lower respiratory tract infections than hrv-a and hrv-b. hrv is the most common cause of upper respiratory tract infections, being responsible for at least % of cases of the common cold. this leads to considerable economic burden in terms of medical visits and both school and work absenteeism. , hrvs have also been linked to lower airway effects that result in significant morbidity and mortality, such as exacerbations of chronic pulmonary disease, severe bronchiolitis in infants and children, as well as fatal pneumonia in elderly and immunocompromised adults. , in general, hrv infections occur during spring and autumn, and manifest differently depending on whether the lower or upper respiratory tract is infected. infections of the upper respiratory tract ordinarily include symptoms of the common cold, but can present as acute otitis media or rhinosinusitis. on the other hand, infections of the lower respiratory tract can cause severe symptoms and result in bronchiolitis and pneumonia. in brazil, since the influenza a pandemic of , referral hospitals have been conducting active surveillance to detect respiratory viruses. such surveillance includes notification and laboratory investigation of cases meeting the diagnostic criteria of severe acute respiratory infection (sari). this viral respiratory infection monitoring has resulted in important information about the circulation of other community-acquired respiratory viruses (crv). the present study reports the epidemiological features, clinical characteristics, and outcomes of hrv infections in comparison with other crv infections in patients hospitalized in a referral hospital in southern brazil, for two consecutive years. patients hospitalized at an academic tertiary care center in southern brazil from whom respiratory samples were collected and sent for investigation, or who were diagnosed with sari in or , were included in the study. respiratory samples (nasal swab or nasopharyngeal aspirate, or bronchoalveolar lavage) were collected in different periods of hospitalization according to medical recommendation. individuals with more than one sample collected during the same symptomatic period were considered as a single case and only the first result was evaluated, so that the number of respiratory viral detections was not overestimated. the medical records and influenza notification forms of patients with detectable respiratory virus were reviewed, focusing on epidemiology, clinical manifestation, outcome, laboratory findings, and diagnosis of sari. sari was defined as a flu-like syndrome with signs of severity (dyspnea or oxygen saturation below %). during the study, a total of cases were identified in both databases. of these, five cases were excluded because the researchers could not access the medical records and a further were excluded as no samples had been sent for virus detection. thus, patients with respiratory samples investigated for respiratory virus detection were included. the institutional ethics review board approved the study (no. # . . . ). rvs were detected using a multiplex reverse transcription polymerase chain reaction (rt-pcr) technique. the viral genome was extracted using a high pure viral rna kit (roche inc., mannheim, germany) in accordance with the manufacturer's instructions. first strand cdna synthesis was achieved using random primers and an improm-ii reverse transcription system (promega inc., wi, usa). the resulting cdna was then subject to pcr by using a seeplex ® rv ace detection kit (seegene inc., korea), in accordance with the manufacturer's protocol. this multiplex pcr technology enables a simultaneous detection of respiratory viruses: human adenovirus (adv), human metapneumovirus (mpv), parainfluenza virus type , , , and (piv- , piv- , piv- , and piv- ), influenza a (flua), influenza b (flub), respiratory syncytial virus type a and b (rsv-a, rsv-b), human rhinovirus types a/b/c (hrv), human enterovirus (ev), human bocavirus (bov), and human coronavirus (cov) types e/nl (alpha coronaviruses) and oc /hku (beta coronaviruses). data were compiled using jmp version . . (sas institute inc., cary, nc, usa) and analyzed using graphpad prism version . (graphpad software inc., ca, usa). parametric and non-parametric tests were used as appropriate. the nonparametric spearman correlation coefficient was used to analyze meteorological data. variables with an associated pvalue < . in the univariate analysis and those considered as confounding factors (age and length of hospitalization) were subjected to multivariate logistic regression to identify independent predictors for severe disease. all p-values were two-tailed and a value of < . was considered significant. curitiba is located in southern brazil and has a temperate climate. data on monthly measures of temperature and rainfall were supplied by the meteorological system of paraná (sistema meteorológico do paraná [simepar]). were compared with those with other crv infections, groups and , respectively) ( table ) . for this analysis, only cases with single infections were included, excluding nosocomial infections, since given that patients with nosocomial infections were already in the hospital, it would be expected that they would have some underlying illness, which could overestimate the severity of infections. most hrv infected patients were younger than years ( %), % were younger than year, and % were younger than months of age. the groups showed no statistically significant difference in median age. gender was unequally distributed between the groups. the main clinical manifestations observed in both groups included fever, cough, and dyspnea. however, the hrv positive group had a significantly lower proportion of cases with fever ( %, p = . ), and more comorbidities than the other group ( % vs. %, p = . ). although immunosuppression, chronic lung disease, and heart disease were the major medical conditions present in both groups, chronic lung disease was more frequent in the hrv group ( %), but without significant difference. concerning the diagnosis of sari, there were fewer patients diagnosed with this syndrome in the hrv group ( %, p = . ) than in the other crv group. in the multivariate analysis of the relationship between clinical characteristics significantly different between the hrv-and other crv-groups, only the higher prevalence of previous comorbidities in the hrv group was significant. patients with no comorbidities were . ( % ci: . --- . ) times more likely to be infected crvs other than hrv, in comparison to patients with comorbidities. despite the fact that most of chest x-rays of hrv infected cases were missed ( %), radiologic alterations were described in % of the performed exams. most of the findings observed were interstitial infiltrate ( %) and pulmonary consolidation ( %), with no significant difference between both groups. severe disease was defined as that requiring mechanical ventilatory support, or intensive care unit (icu) admission, or death during hospitalization; no significant difference was found between the groups. two hrv infected patients died during hospitalization, both from respiratory infection complications. hrv had the highest rates of coinfection ( %) of the samples with more than one crv detected. the viruses most co-detected with hrv were ev ( / ; %) and rsv ( / ; %), as shown in fig. . in % of cases, the co-detection involved hrv plus another virus, and in % of cases, hrv was associated with two or more viruses. in cases, hrv was the only virus detected. a comparison of the clinical characteristics of monoinfected hrv patients with co-infected hrv patients was performed, and no relation between viral co-detection and disease severity (p = . ) was found. the clinical features significantly associated with hrv co-detection were as follows: age --- months (or = . ; % ci: . --- . ), length of hospitalization more than days (or = . ; % ci: . --- . ), and no sari diagnosis (or = . ; % ci: . --- . ). as co-infection with hrv alone was not related with severe disease, a second analysis was performed comparing disease severity between hrv co-infected patients and patients infected with other crvs. neither the presence nor absence of hrv co-detection was associated with disease severity (p = . ). cases of hrv infection occurred throughout the study period (fig. ) . the months of may to august demonstrated the highest number of hrv infections during , peaking in august ( cases; %). in , the seasonality of hrv infection was from march to may, peaking in may ( cases; %). comparing the monthly distribution of hrv cases with average temperature ( • c) and rainfall (mm), a negative correlation between the number of hrv cases and the average temperature (rs = − . , p < . ) was demonstrated, but there was no significant correlation with rainfall (rs = − . , p = . ). since the implementation of systematic investigation into respiratory viruses in hospitalized patients with sari, hrv has been found with high frequency, either alone or codetected with other respiratory viruses. since some of these infections may have a poor prognosis, including death, it is critical to know and better characterize this infection to be prepared for its early diagnosis, appropriate clinical management, and prevention of nosocomial spread. the high frequency of hrv found in clinical samples from patients with sari was similar to that reported by kim et al. in a tertiary hospital in korea. he et al. analyzed a hospitalized pediatric population and found a prevalence of %, while walker and ison found a prevalence of % in a hospitalized adult population. this difference between age groups was observed in this study, emphasizing the greater vulnerability of children to this infection, probably as consequence of a more intense inflammatory process triggered by primary infection or by favorable anatomical conditions of the respiratory tract in younger children. rsv was the first pathogen identified to be associated with the severe respiratory disease in children. however, since the development of molecular techniques to detect hrv, this virus has been the focus of the most recent studies in this field. nowadays, hrv infections have been linked with exacerbations of chronic lung diseases and fatal pneumonia in patients at the extremes of age or with pre-existing comorbidities. , the importance of one study that evaluated the characteristics of hrv infections in a tertiary hospital is based on these new concerns. marcone et al. reported a higher number of hrv infections in hospitalized children in argentina when compared with pediatric outpatients with acute respiratory infection, demonstrating a scenario that contributed to the increased concern about hrv infection in the hospital setting. previous studies have reported that the hrv infection rate in hospitals varies from % to %. , , however, this variation is probably accounted for by the differences in age and clinical characteristics of patients analyzed in each study. although this study included all age groups, a predominance of pediatric patients infected with hrv was observed, mostly younger than year old. in addition, the hrv-positive group had a greater proportion of patients with pre-existing comorbidities than the group with other identified crvs. , , --- among the comorbidities observed in this study, chronic lung diseases were frequent in the hrv group, a correlation that has already been demonstrated in another similar study, which corroborates the close association of hrv infection and exacerbations of chronic lung diseases, such as chronic obstructive pulmonary disease (copd), asthma, and cystic fibrosis. of samples positive for crvs, samples ( %) had at least two co-detected viruses, in keeping with the expected --- % encountered in hospitalized individuals. , , the viruses most frequently co-detected with hrv were ev ( %), rsv ( %), and adv ( %); consistent with previously reported patterns. , however, the high frequency of ev and hrv co-detection may be as a result of using the nrt region of the viral genome to identify both pathogens, which may have lowered the specificity of the rt-pcr test. a more appropriate means to discern between these species would be to carry out the rt-pcr followed by nucleotide sequencing of amplicons. the high level of dual infection involving hrv and rsv is often explained both by the coexistence of these viruses throughout the year and their similar seasonal variation, but this hypothesis is not unanimous. in contrast to findings reported by goka et al., this study did not show an association between disease severity and viral co-detections. furthermore, hrv was not found to have a protective influence in cases where it was involved in codetection, as there was no significant difference in disease severity when comparing the groups with or without hrv co-detection. likewise, in contradiction to the present findings, asner et al. observed an increased disease severity in children infected with hrv alone. among the probable factors associated with these contrasting findings, the following may be cited: (i) analysis in different groups of patients (outpatient and hospitalized), (ii) assessing a small number of patients, and (iii) adoption of different severity criteria. fever was less frequent in patients infected with hrv compared to other crvs. , chest x-ray findings were normal in %, showed interstitial infiltrate in %, and demonstrated pulmonary consolidation in % of cases, with no significant difference between the hrv and other crv groups. these patterns are similar to those reported by fica et al. in hospitalized adults in chile. there were significantly fewer cases of sari diagnosed in the patients infected with hrv than other crvs. overall, % ( / ) of patients with sari were infected with hrv. this concurs with a previous study. reports about the seasonality of hrv infection, including a study from argentina, which is in close geographical proximity of southern brazil, show that it circulates mainly in autumn and spring. , , although hrv occurred in almost all months of the study period, different peaks were observed in and , and neither peak included spring. in , the highest prevalence of hrv infection was in autumn, followed by winter, and vice versa in . the analysis of meteorological data found a negative correlation between the number of hrv cases and the average temperature, but no significant correlation with rainfall. however, in order to more accurately establish the seasonality of hrv infections, the analysis should include additional years. this research had some limitations: (i) it was a retrospective study and some medical records were incomplete; (ii) hrv species were not identified, which would have yielded important data since the genotypes reportedly have different virulence; (iii) this analysis was carried out only with hospitalized patients, which may have overestimated the impact of hrv infection; and (iv) it was not possible to evaluate the frequency of nosocomial hrv infection, data critical to guide preventive measures in the most affected settings. however, this is the first report about hrv infections in the region and a critical analysis of the data is important to obtain greater awareness of the dynamics of dispersion and impact of these respiratory viruses in the community. in conclusion, hrv has a high prevalence in the hospitalized children and was present in cases of severe disease, including death. however, a dependent relationship between the presence of hrv in viral co-detections and severity of disease was not observed in this study. conflicts in the literature demand a closer analysis of cases of co-detection involving hrv with review of the data to determine the impact of this, since the lack of standardization between studies probably contributes to the divergent data. hrv infection is closely associated with comorbidities, mainly chronic lung diseases, and is an important factor in exacerbations of these underlying lung diseases. the colder seasons were the period with higher frequency of hrv infections in southern brazil, and therefore must be a period to warn clinicians about young age children affected by respiratory infections, especially those with comorbidities such as chronic lung disease. patients with this profile should have respiratory samples collected to identify possible viral infection, and if hrv is detected, the medical staff should be ready for adequate management, taking into consideration the possible poor outcomes. smr is sponsored by a cnpq fellowship. the authors declare no conflicts of interest. analysis of the complete genome sequences of human rhinovirus the abcs of rhinoviruses, wheezing, and asthma clinical and molecular features of human rhinovirus c human rhinoviruses wheezing rhinovirus illnesses in early life predict asthma development in high-risk children seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center epidemiology of respiratory viral infection using multiplex rt-pcr in a -year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china. influenza other respir viruses respiratory viral infections among hospitalized adults: experience of a single tertiary healthcare hospital. influenza other respir viruses genetic diversity and clinical impact of human rhinoviruses in hospitalized and outpatient children with acute respiratory infection, argentina clinical relevance of rhinovirus infections among adult hospitalized patients do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? respiratory viral coinfections identified by a -plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features single, dual and multiple respiratory virus infections and risk of hospitalization and mortality evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children rhinovirus detection using different pcr-based strategies is virus coinfection a predictor of severity in children with viral respiratory infections? rhinovirus is an important pathogen in upper and lower respiratory tract infections in mexican children epidemiology of pathogen-specific respiratory infections among three us populations key: cord- -hcpa ej authors: renwick, neil; schweiger, brunhilde; kapoor, vishal; liu, zhiqiang; villari, joseph; bullmann, reinhard; miething, robert; briese, thomas; lipkin, w. ian title: a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: hcpa ej acute respiratory infection is a significant cause of morbidity and mortality in children worldwide. accurate identification of causative agents is critical to case management and to prioritization in vaccine development. sensitive multiplex diagnostics provide us with an opportunity to investigate the relative contributions of individual agents andmayalso facilitate the discovery of new pathogens. recently, application of masstag polymerase chain reaction (pcr) to undiagnosed infuenza-like illness in new york state led to the discovery of a novel rhinovirus genotype. here we report the investigation, by masstag pcr, of pediatric respiratory-tract infections in germany, studying cases for which no pathogen was identified through routine laboratory evaluation. respiratory viruses were identified in cases ( %); of the identified viruses, ( %) were rhinoviruses. the novel genotype represented % of rhinoviruses and % of all identified viruses. infections with the novel genotype were associated with upper-respiratory-tract symptoms but, more frequently, with bronchitis, bronchiolitis, and pneumonia. human rhinoviruses (hrvs) are the most frequent cause of acute respiratory illness worldwide. although hrvs are most commonly associated with mild upperrespiratory-tract disease [ ] [ ] [ ] , infection of lower airways does occur [ - ] . lower-respiratory-tract infections (lrtis) related to hrv are increasingly being reported in infants, elderly persons, and immunocompromised patients [ ] . hrvs are also implicated in exacerbations of asthma [ , ] , chronic bronchitis [ ] , and acute bronchiolitis [ ] . taxonomically, hrvs are currently grouped into species, human rhinovirus a (hrv-a) and human rhi-novirus b (hrv-b), in the genus rhinovirus of the family picornaviridae ( [ , ] ). these nonenveloped, positive-sense, single-stranded rna viruses have been classified serologically [ , ] and on the basis of their antiviral susceptibility profiles [ , ] , their nucleotidesequence relatedness [ , ] , and their use of receptors (intercellular adhesion molecule , low-density lipoprotein receptor, and decay-accelerating factor) [ ] [ ] [ ] . phylogenetic analyses of the vp /vp and vp coding regions have indicated the presence of serotypes in genetic group a and serotypes genetic group b [ - , ]. an agent is commonly not implicated in up to % of cases of severe respiratory disease, despite the application of polymerase-chain-reaction (pcr) assays as well as classical diagnostic methods, including antigen tests, serology, and culture methods. broad-range molecular assay systems, such as multiplex pcr (hexaplex [ ] , genescan [ ] , and masstag [ ] ), or microarrays (vi-rochip [ ] and panmicrobial greenechips [ ] ) may therefore allow us to gain new insights into epidemiology and clinical associations [ , ] . with respect to hrv, recent studies employing sensitive pcr systems for these difficult-to-isolate organisms have shown an increased detection rate, compared with culture methods [ , [ ] [ ] [ ] [ ] . applying a multiplex masstag pcr platform, we recently detected numerous agents of respiratory illness in samples that had been submitted for laboratory diagnosis but that had tested negative during routine diagnostic assessment [ ] . hrvs were identified at high frequency in this set of samples. detailed genetic analysis indicated that a large fraction of these viruses represent a previously uncharacterized genotype of rhinovirus, one that diverges from either hrv-a or hrv-b. in an attempt to gather additional information on the potential pathogenicity, as well as temporal and geographic distribution, of rhinoviruses, including the recently identified genotype, we evaluated specimens collected, during the - seasons in bad kreuznach, germany, from children hospitalized because of severe lrti. nasopharyngeal aspirates were obtained from children admitted, because of acute respiratory-tract infection, to the kreuznacher diakonie hospital (bad kreuznach, germany) during the interval of - . individuals ranged in age from weeks to years (mean age, months; median age, months); % were male, % female. specimens collected at the time of admission were forwarded undiluted to the robert koch institute (berlin, germany) for laboratory evaluation. rna extraction was performed by use of qiaamp viral rna kits (qiagen). the samples for which no pathogen had been diagnosed after assessment by realtime reverse-transcription (rt)-pcr assay for influenza virus [ ] and respiratory syncytial virus infection were stored at assay procedures. the rna samples representing cases of undiagnosed respiratory diseases were employed as a template for cdna synthesis by use of superscript ii kits with random hexamer priming (invitrogen), and they were analyzed by masstag pcr by using a viral primer panel [ ] that targeted influenza virus a and b (fluav and flubv, respectively), respiratory syncytial virus a and b (rsv-a and rsv-b, respectively), human parainfluenza virus , , , and (hpiv- , hpiv- , hpiv- , and hpiv- , respectively), human coronavirus e and oc- (hcov- e and hcov-oc , respectively), human metapneumovirus, entero-and rhinoviruses, and adenoviruses. the fidelity of the masstag pcr signal was verified by reamplification of products and by sequence analysis for all positive specimens. in instances in which masstag pcr indicated the presence of a picornavirus, the vp /vp region was amplified [ ] . amplification products were purified from agarose gels (qia gel extraction kit; qiagen), and nucleotidesequencing reactions were performed on both strands by use of the abi prism big dye cycle sequencing kits and the abi prism genetic analyzer systems (applied biosytems). identical results were obtained with duplicate aliquots processed at the new york and berlin laboratories. sequence analyses, alignments, and phylogenetic reconstructions were performed by use of programs from the wisconsin gcg package (accelrys) and by mega . software [ ] . nucleic-acid sequences generated during this work are available at genbank, under the accession numbers eu -eu . we used masstag pcr to investigate nasopharyngeal aspirates from children hospitalized because of acute respiratory illness for which no pathogen was identified through routine laboratory testing. masstag pcr identified at least candidate respiratory-viral pathogen in specimens. although there was variability across the seasons clinical associations. hrvs were the viruses most frequently detected in our set of samples, representing % ( / ) of the identified viruses; coinfection with another virus was observed in only % ( / ) of these cases (table ). the frequency of fever or cough in infections with hrv ( %) was comparable to that in infections with the other viruses ( %); the frequency of rhinitis or pharyngitis with hrv ( %) was comparable to that with other viruses; and the frequency of lrti symptoms (bronchitis, bronchiolitis, and pneumonia) with hrv ( %) was comparable to that with the other viruses ( %). whereas pneumonia was more common in infections with hrv a/b ( %) than in infections with hrv x ( %), the frequency of bronchiolitis with hrv a/b ( %) was comparable to that with hrv x ( %), and the frequency of bronchitis with hrv a/b ( %) was comparable to that with hrv x ( %). lrti was recorded in % of hrv x infections; however, some cases were related to milder disease (table ) . molecular epidemiology of identified picornaviruses. masstag pcr targets conserved sequences in the 'untranslated region of entero-and rhinoviruses; thus, to facilitate phylogenetic analysis of hev and hrv, we amplified and sequenced the vp /vp gene region. however, when we used the basic local alignment search tool for analysis at the nucleotide level, we did not find, for of the vp /vp sequences, a significant match with hrv-a, hrv-b, or hev sequences; analysis at the amino-acid level revealed homology to enteroand rhinoviral sequences, with a sequence identity of %- %. high similarity at both the nucleotide-and amino-acid levels was evident when sequences were aligned with an unclassified genetic clade of picornaviruses recently identified in new york state [ ] . however, detailed phylogenetic analysis indicated significant sequence diversity among the viruses ( in this study of samples collected, during a -year interval, from hospitalized children with severe undiagnosed respiratory infection, masstag pcr allowed us to detect viral pathogens in ( %) of cases. the pathogens most commonly identified were hrvs. these findings are consistent with other studies, which have indicated that rhinoviruses or picornaviruses ac-count for %- % of acute respiratory infections [ , , , - ]-exceeding, in some instances, even the frequency of rsv infection in pediatric-patient populations [ , [ ] [ ] [ ] . the presence of hrv is not sufficient to prove causation. asymptomatic hrv infection has been described; however, the extent to which infection without disease represents carriage, incubation, or convalescence is unknown [ , , , , ] . although we did not have samples to test for the presence of hrv in the lower respiratory tract, the high frequency at which hrv was identified as being the sole virus detected suggests a correlation between the agent and the observed lrti symptoms. support for the plausibility of hrv being pathogenic in lrti comes from the facts that ( ) in situ hybridization has demonstrated that hrvs exist in lower airways and ( ) hrvs have been shown to trigger inflammatory processes in the infected cells and tissues [ ] [ ] [ ] [ ] [ ] [ ] [ ] . among the hrvs identified in the present study were representatives of the novel genetic clade recently discovered in new york state [ ] ; indeed, these viruses comprised the majority of hrvs detected. hrv-a and hrv-b have been implicated in common colds as well as in severe lrti. in our patients, viruses of the novel genetic clade were also associated with a wide range of diseases, ranging from rhinitis to bronchitis to severe pneumonia, necessitating supplemental oxygen in ϳ % of cases. a seasonal pattern of hrv infections has been described [ , , ] ; however, data regarding ( ) pathogens detected (no.) hpiv- ( ) rsv-b ( ) hpiv- ( ) hadv ( ) hpiv- ( ) hpiv- ( ) hev/hrv ( ) hpiv- ( ) hmpv ( ) hcov-oc ( ) hev/hrv ( ) hadv ( ) hev/hrv ( ) specific identification (no. either serotype-or genotype-specific patterns of seasonality or disease symptoms are limited [ - ] . a temporal trend of sequence diversity or of correlation between genotype (within the novel hrv clade) and clinical diagnosis was not apparent in our data (figure ). no detailed information is available yet concerning the history of the novel hrv clade; nonetheless, the sequence diversity observed within it (figure ) is not consistent with a re-cent introduction. this clade may account, in part, for earlier reports of nontypeable rhinoviruses [ , ] . indeed, its discovery may reflect the implementation of new technologies rather than novelty of the agent itself. we anticipate that future work will define more than serogroup. our findings reinforce other groups' recent work indicating the significance of hrvs in pediatric lrti. the presence of novel hrvs in disparate geographic locations, in association with seri- ous respiratory disease in children as well as in adults, mandates further work in epidemiology and pathogenesis. frequency and natural history of rhinovirus infections in adults during autumn viruses and bacteria in the etiology of the common cold the seasonality of rhinovirus infections and its implications for clinical recognition characterization and classification of echo -rhinovirus-coryzavirus agents detection of rhinovirus rna in lower airway cells during experimentally induced infection rhinoviruses infect the lower airways quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinovirus and the lower respiratory tract interleukin- gene expression in acute virus-induced asthma rhinovirus viremia in children with respiratory infections viral infections of human association of rhinovirus infection with increased disease severity in acute bronchiolitis international committee on taxonomy of viruses. universal database of the international committee on taxonomy of viruses virus taxonomy: eighth report of the international committee on taxonomy of viruses antigenic groupings of rhinovirus serotypes a collaborative report: rhinoviruses-extension of the numbering system from to two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity alignment of capsid protein vp sequences of all human rhinovirus prototype strains: conserved motifs and functional domains molecular relationships between human rhinovirus serotypes genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus many rhinovirus serotypes share the same cellular receptor the major and minor group receptor families contain all but one human rhinovirus serotype human rhinovirus and enterovirus represent a unique serotype with rhinovirus and enterovirus features vp sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types , , and by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness diagnostic system for rapid and sensitive differential detection of pathogens microarray-based detection and genotyping of viral pathogens panmicrobial oligonucleotide array for diagnosis of infectious diseases masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influeza-like illness in new york state during microarray detection of human parainfluenzavirus infection associated with respiratory failure in an immunocompetent adult improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr molecular diagnosis of human rhinovirus infections: comparison with virus isolation detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays mega : integrated software for molecular evolutionary genetics analysis and sequence alignment use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms novel rhinovirus genotype in lrti • jid respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care: ige and eosinophil analyses rhinovirus-associated hospitalizations in young children persistence of rhinovirus and enterovirus rna after acute respiratory illness in children lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects low grade rhinovirus infection induces a prolonged release of il- in pulmonary epithelium rhinovirus replication causes rantes production in primary bronchial epithelial cells rhinovirus infection increases -lipoxygenase and cyclooxygenase- in bronchial biopsy specimens from nonatopic subjects rhinovirus infections in an industrial population. v. change in distribution of serotypes the seattle virus watch. v. epidemiologic observations of rhinovirus infections, - , in families with young children rhinoviruses in britain - we thank ingrid zadow, bettina bauer, manuela friedrich, marlies hartwig, and sven tietze for their excellent technical assistance. key: cord- -wyzb v a authors: forsyth, m.; al-nakib, w.; chadwick, p.; stanway, g.; hughes, p. j.; leckie, g.; almond, j. w.; tyrrell, d. a. j. title: rhinovirus detection using probes from the ′ and ′ end of the genome date: journal: arch virol doi: . /bf sha: doc_id: cord_uid: wyzb v a this study investigated the abilities of cdna probes from the ′ and ′ ends of the genome of human rhinoviruses (hrv-) , , and b to detect rna from rhinovirus serotypes. the results show that probes from the ′ end of the genomes of hrv- , , and b detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. in contrast, all the ′ end probes were specific for the homologous virus. however, along hrv- probe detected a large number of serotypes. it was concluded that such cdna probes would not detect all serotypes with equal efficiency. synthetic oligonucleotides corresponding to short but highly conserved regions in the ′ non coding region may overcome this problem. rhinoviruses are the major causative agents of the common cold [ ] . in the majority of healthy individuals, the infection results in a short illness of some - days duration characterized by rhinorrhoea, nasal obstruction, sore throat and pharyngitis [ ] . however, in immunocompromised individuals particularly children and in patients with obstructive airways disease, rhinovirus infection may result in more serious lower respiratory tract involvement [ , ] . furthermore, recent community studies in michigan, u.s.a., suggested that rhinoviruses can be isolated from up to percent of adults (over years of age) with lower respiratory tract involvement [ ] . in these individuals the median duration of illness was as long as weeks [ ] . we have recently shown that a new synthetic anti-rhinovirus agent, r , can sucessfully suppress illness in volunteers challenged with a rhinovirus [ ] . it is anticipated that with further progress in the field it may be possible to treat these infections. however, rapid virus identification prior to treatment would be essential since these antivirals are specific for rhinoviruses. until recently, rhinoviruses could only be identified by growth in a sensitive cell culture. such procedures are time consuming, labour intensive and require considerable expertise. although, it is now possible to detect rhinovirus antigens directly in nasal washings using immunologically based methods such as elisa [ ] , the diversity of serotypes, recently estimated to be around [ ] makes efficient detection of all serotypes difficult. we have therefore, attempted to overcome this problem by developing procedures based on r n a detection. a previous study has shown that a c d n a probe from the ' non-coding region of hrv- detects . % of the rhinoviruses investigated, but the sensitivity of detection was variable and presumably depended on the degree of genomic homology of particular serotypes with hrv- [ ] . in this study we therefore increased the number of probes used to include the ' ends of hrv- , b and and the ' ends of h r v - b [ ] and [leckie et al., in prep.] . the aim was to find a probe that would hybridise with r n a from various serotypes with equal efficiency and we therefore studied the reactions of the new probes with r n a from rhinovirus serotypes. stocks of rhinoviruses including animal rhinoviruses such as the calf rhinovirus sdi and bovine rhinovirus ec and other control viruses, namely influenza a and b (flu a and b), coronavirus e and coxsackie a (coxa ) were prepared as previously described [ ] . each stock was titrated in microtitre plates and titres expressed as tcids /ml. table shows the final titres of the virus stocks as used in this study. viral rna was extracted using the method of rotbart et al. [ , ] . briefly, . ml of each virus preparation was mixed with an equal volume of a : mixture of x ssc-- % formaldehyde. the mixture was then spotted onto nitrocellulose filters that had been pre-soaked in x ssc as described earlier [ ] . details of the methods used to prepare and label probes used in this study have been described previously [ , ] . briefly, probes were produced from m templates containing rhinovirus cdna cloned in the appropriate orientation. the hrv- ' construct contained nucleotides - and was produced by cutting a recombinant plasmid with psti and bglii and ligating into the m mpl psti and bamhi sites. the corresponding ' probe comprised positions - contained within an hpai fragment ligated into m cut with sinai. the hrv- b probes represented positions - ( ') located within a psti hindiii fragment which was ligated into these sites of m and - ( ') located in a psti fragment. the hrv- ' end probe was prepared from a . kb cdna clone of hrv- designated prg . a base pair fragment representing nucleotides - of the hrv- sequence (unpublished) was subcloned into m mp in the positive sense orientation. both of the ' end probes used in this study were prepared from a . kb hrv- cdna clone pr covering the ' region of the genome. the short ' end probe was prepared from a base pair fragment representing nucleotides - . the total length of the hrv- genome is excluding poly a tail. the long ' end probe was prepared from a base pair fragment from pr covering nucleotides - . in both cases the fragments were subcloned into the phage vector m mp in the positive sense orientation. the templates were used to produce radioactive cdna probes complementary to viral sense rna by extension of an m universal primer in a reaction performed by the klenow fragment of dna polymerase . annealing of the primer/template was achieved by boiling together ( min) in a mixture ( lal) comprising primer ( ng), template ( lag), mm tris-hc , ph . , and . mm mgci . the solution was allowed to cool to room temperature and to it was added dgtp, dctp dttp (to a final concentration of . ram), p_ datp ( laci) klenow fragment ( units) and water to give a final volume of ~tl. the reaction took place at room temperature for minutes after which the radioactive dna was separated from unincorporated nucleotides by passage through a sephadex g column. the probes were hybridized with viral rna as described previously [ , ] . the strength of the hybridization signals was assessed visually by two independent observes and the signal was classified as very strong (+ + + +), strong (+ + + ), good (+ +), positive (+), weak ( -), or no signal ( ). comparison of the hybridization results with the titres of the virus stocks (table ) shows that generally there is no direct relationship between the titres and the strength of the hybridization reaction. for example, the hrv- probe hybridised very strongly (+ + + +) with hrv- even though the titre was low (< tcids /ml) but only weakly with hrv- a ( + ) which had one of the highest titres of the viruses tested (> tcids /ml) ( table ) . it therefore appears that the efficiency of detection is more directly related to other factors, the most important of which is probably the degree of rna homology between the rna of the different rhinoviruses. table shows the strength of the signal observed using the various viruses and seven probes. it can be seen that the reactions varied greatly in intensity. thus, the hrv- , ' end probe gave a very strong signal (+ + + +) when hybridized with rna from hrv- , , and and a strong signal (+ + +) when reacted with rna from hrv- , , , , , , and . similarly, a ' end probe from hrv- gave a very strong signal (+ + + +) with rna from hrv- , , and . the ' end probe from hrv- b hybridized very strongly (signal + + + + ) with rna from hrv- and and strongly (+ + + ) with rna from hrv- a, , and . in contrast to the ' end probes, those from the ' end ( , , and nucleofides in length for hrv- , b, and , respectively) detected only the homologous virus in the conditions of the assay. however, the longer probe ( nucleofides in length) from hrv- , detected many more viruses (table ) . indeed, this probe hybridized very strongly (+ + + + ) with rna from hrv- , , , , , , and and reacted strongly (signal + + +) with rna from hrv- , , , , and suggesting that these viruses are closely related to hrv- in the ' end region of the genome and perhaps reflects the conservation of the polymerase sequences among these viruses. both ' and ' end probes from hrv- , - , and - b reacted with their respective viruses very strongly (+ + + + ). table shows the percentage of rhinovirus serotypes detected by each probe according to the strength o f the hybridization signal. thus . , , and . % of viruses investigated were detected (signal > + ) by hrv- , , and b, ' end probe, respectively, while % were detected by the long ' end hrv- probe. similarly, . , . , and . % of rhinoviruses gave a good hybridization signal ( > + + ) with ' end probes from hrv- , , and b, respectively, while . % gave a similar signal with the long hrv- , ' end probe. as can be seen from table , none of the control respiratory viruses such as influenza a, b, and coronavirus e, gave positive hybridization signals in any experiments during this study. coxsackie a , gave a positive signal with hrv- ' end probe suggesting some genomic homology with hrv- . these hybridization tests were repeated three or more times and the results were shown to be reproducible. the data suggest that c d n a hybridization with different probes show a different relationship between rhinovirus serotypes from that based an other properties. for example, hrv- which shares the same cellular receptor as hrv- (both are included in the major receptor group) [ , ] reacted more strongly (signal + + + ) with the ' end probe from hrv- b, a serotype in the minor receptor group than with hrv- (signal +). similarly, hrv- which shares the same cellular receptor as hrv- b (both are included in the minor receptor group) [ , ] reacted more strongly (signal + + + ) with the ' end probe from hrv- , a serotype from the major receptor group, than with hrv- b (signal +). moreover, this relationship is also different from that based antigenic crossreactivity [ ] . it is interesting to note that r n a from hrv- hybridized extremely well (signal + + + +) with both hrv- and b ' end probes suggesting a strong genomic homology between hrv- and these two viruses. similarly, r n a from hrv- reacted very well with the ' end probe from both hrv- and b indicating a close genomic relationship between hrv- and these two serotypes in the ' end. furthermore, rna from hrv- and hybridized extremely well with both ' and ' end probes from hrv- implying that these viruses have strong genomic homology with hrv- in both ends of the genome. in contrast, rna from hrv- , t, , and (signal < +) and and (signal + to -) did not hybridize well with any of the probes investigated probably indicating that these viruses are more divergent from hrv- , , and lb. the findings of this study are that probes from the ' end of the genome of rhinoviruses detect a large number of rhinoviruses, although the detection rate is variable and apparently depends on the strength of genornic homology among the different serotypes. in contrast, probes from the ' end of the genome (of some nucleotides) of hrv- and b detected only the homologous virus under the hybridization conditions of the assay. however, a similar size probe from the ' end of hrv- detected many more serotypes. in contrast, a shorter probe ( nucleotides in length) also from the ' end of hrv- , detected only the homologous virus, thus indicating that the detection rate is highly influenced by probe length. both ' and ' end probes detected the homologous viruses with equal efficiency. it was interesting to note that the ' end hrv- probe was more efficient than the other probes in detecting a larger number of rhinoviruses. this is somewhat surprising since comparative sequence analysis indicates that hrv- is relatively diverse from the majority of rhinoviruses studied. it might therefore be thought that probes from hrv- b and hrv- , which are more typical rhinoviruses, would prove to give a higher detection rate. the data presented in this paper are interesting in that they show that cdna probes are unlikely to be useful in detecting all rhinoviruses with equal efficiency despite an earlier prediction that the ' end non-coding region was likely to be relatively highly conserved throughout the rhinovirus genus [ ] . these results are therefore in agreement with our earlier findings with the hrv- probe [ ] and show that although there is considerable homology in the ' end noncoding region of many rhinoviruses it is still not sufficient for a probe prepared from this region to detect all the different rhinovirus serotypes with equal efficiency. furthermore, in tests on clinical material, both the identity of the infecting serotype and its concentration in nasal secretion would vary widely. in this study there were great variations in the signal given by the different serotypes even though the titres of virus used were usually greater than tcids /ml which is much higher than that normally found in nasal washing (often < l tcids /ml). recent work with synthetic oligonucleotides corresponding to short but highly conserved regions in the ' end non-coding region of the rhinovirus genome [ ] suggests that such probes will detect all rhinovirus serotypes with equal efficiency [ ] . further studies are in progress. many rhinovirus serotypes share the same cellular receptor detection of human rhinoviruses and their molecular relationship using cdna probes rhinovirus detection by cdna: rna hybridization common cold viruses--rhinoviruses suppression of colds in human volunteers challenged with rhinoviruses by a new synthetic drug (r ) synthetic oligonucleotides as diagnostic probes for rhinoviruses isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses antigenic grouping ofg rhinovirus serotypes the common cold: control? direct detection of rhinoviruses by an enzyme-linked immunosorbent assay provocation of airflow limitation by viral infection: implication for treatment a collaborative report: rhinovirus---extension of the numbering system from to the nucleotide sequence of human rhinovirus b: molecular relationships within the rhinovirus genus the association of rhinoviruses with lower respiratory tract disease in hospitalized patients rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes use of subgenomic poliovirus dna hybridization probes to detect the major subgroups of enteroviruses factors affecting the detection of enteroviruses in cerebrospinal fluid with coxsackievirus b and poliovirus i cdna probes rhinovirus detection using cdna probes the complete nucleotide sequence of common cold virus: human rhinovirus dr. glyn stanway is supported by a grant from the medical research council number g ca. key: cord- - yvs a authors: han, tae-hee; chung, ju-young; hwang, eung-soo; koo, ja-wook title: detection of human rhinovirus c in children with acute lower respiratory tract infections in south korea date: - - journal: arch virol doi: . /s - - -z sha: doc_id: cord_uid: yvs a recently, hrv-c was identified as a new species of hrv, but its spectrum of clinical disease is still not clear. the purpose of this study was to investigate the molecular epidemiology of hrvs in children with acute lower respiratory tract infections (lrtis). a total of hrv-positive samples that were negative for other respiratory viruses were sequenced. hrv-a was detected in , hrv-b in , and hrv-c in of these samples. all hrv-c-positive patients showed favorable clinical outcomes. we confirmed the presence of hrv-c in children with lrtis, but its association with clinical severity is not clear. human rhinoviruses (hrvs) are the most frequent cause of acute respiratory illness worldwide [ ] [ ] [ ] [ ] [ ] . although hrvs are most commonly associated with mild upper respiratory tract disease, infection of lower airways does occur [ , ] . lower respiratory tract infections (lrtis), especially in infants, the elderly, and immunocompromised patients are increasingly being reported [ ] [ ] [ ] . hrvs are currently classified into two species, hrv-a and hrv-b, in the genus rhinovirus of the family picornaviridae [ ] . phylogenetic analysis of the vp /vp and vp coding regions indicated the presence of serotypes in genetic group a and serotypes in genetic group b [ , ] . in recent studies, a member of a newly identified species, hrv-c, has been suggested as an etiologic agent in children with acute respiratory disease such as bronchiolitis, pneumonia, and asthma exacerbation [ ] [ ] [ ] [ ] [ ] . the purpose of this study was to investigate the molecular epidemiology of hrvs in children hospitalized with acute lrtis in south korea. from january to december , a total of nasopharyngeal aspirates were collected from hospitalized children (male/female, / ; median age, months; range of age, - months) with acute lrtis at sanggye-paik hospital, seoul, south korea. all specimens were tested for the presence of human respiratory syncytial virus (hrsv), influenza virus a, influenza virus b, parainfluenzavirus, adenovirus, human metapneumovirus (hmpv), human bocavirus (hbov), and human coronaviruses (- e, -oc , -hku- , and -nl ) by rt-pcr, as described in our previous study [ ] . from the hrv-positive samples, a total of samples that were negative for other respiratory viruses were included in this study for subsequent sequence analysis. viral rna was extracted from each sample using a qiaamp viral mini kit (qiagen gmbh, hilden, germany) according to the manufacturer's instructions. rna was quantitated using nanodrop (thermo scientific, wilmington, de, usa). reverse transcription was performed on . lg of . after incubation at °c for h, the samples were heated for min at °c to stop the reaction. semi-nested pcr for amplification of * bp of the noncoding region of hrvs from clinical specimens was performed. primer p - (caagcac ttctgtywcccc nt - , reference strain l ) was used as the forward primer, and multiple primers were used as reverse primers: p - (acggacacccaaagtag, nt - ), p - (ttagccacattcaggggc, nt - ), p - (ttagccacattcaggagcc, nt - ) and p - (ttagccgcattcagggg, nt - ), as described previously [ ] . a first round of pcr was performed on the samples using p - and p - , followed by a second round of pcr using p - , p - , p - , and p - . pcr was done using the following reaction conditions: initial denaturation at °c for min; cycles of °c for s, °c for s, and °c for min; and final extension at °c for min. in the pcr reaction, a forward primer ( accractactttgggtgtccgtgt , position - in hrv-c ) and a reverse primer ( tciggiadyttccaicaccaicc , position - in hrv-c ) were used to generate a * -bp pcr product encompassing a portion of the untranslated region, the full viral capsid protein (vp) gene, and a portion of the vp gene of the hrv genome. the pcr was done using the following reaction conditions: initial denaturation at °c, min; cycles of °c for s, °c for s, and °c for min; and final extension at °c for min. the amplicon was purified using a qiaquick kit (qiagen gmbh, hilden, germany) and sequenced in both directions using a bigdye terminator v . cycle sequencing kit (applied biosystems, foster city, ca, usa). sequencing products were resolved using an abi xl autoanalyzer (applied biosystems, foster city, ca, usa). nucleotides sequences were aligned using bioedit v . and presented in a topology tree, prepared in mega . [ ] . partial ncr sequences (* bp, nt - of genbank accession no. l ) for the hrv strain were submitted to genbank (fj - , fj - ) . a total of single hrv-positive specimens from children hospitalized with acute lrtis were sequenced after performing rt-pcr based on the ncr region. phylogenetic analysis based on -ncr gene analysis showed that of the hrv strains were hrv-a, were hrv-b, were hrv-c, and the species was undetermined for ( fig. ) . rt-pcr assays based on the vp /vp region were performed to determine the species, and phylogenetic analysis was possible with specimens, which showed that were hrv-a, were hrv-b, and were hrv-c. nine strains for which the species was not determined from the noncoding region belonged to hrv-a in cases and hrv-b in (fig. ) . hrv-c was detected in patients ( boys and girls, months to months of age (mean age months, median age months)) and the diagnoses were asthma exacerbation in patients, bronchiolitis in , and pneumonia in . none of the hrv-c-positive patients required admission to the intensive care unit, and their clinical outcomes were favorable. hrv-c was detected mostly in the spring, while hrv-a showed a peak in september . co-circulation of hrv-a and hrv-c was noted in spring and autumn. to our knowledge, this is the first study to confirm the presence of hrv c infection in children with acute lrtis in korea. recently, novel hrv species were identified and their members were reported to be associated with acute respiratory tract infections with febrile wheeze, asthmatic exacerbation, influenza-like illness, pneumonia and rhinitis [ , , , ] . although several novel hrv species have been identified due to the development of molecular methodology, it is difficult to compare these novel hrvs because different regions of the genome have been used for analysis. in recent studies [ , ] , molecular typing of rhinovirus using the -ncr region has been suggested to be a simple and reliable method for classifying hrv serotypes, because analysis of the vp- or vp -vp region requires multiple primer pairs for rt-pcr. an association of members of novel hrv species with severe respiratory tract infections [ , ] and a global distribution of members of novel species in respiratory specimens have fig. phylogenetic tree of clinical viral isolates (n = ) based on analysis of * bp from the noncoding region (nt - of hrv l ). the phylogenetic tree was built using the neighborjoining method with the kimura two-parameter estimation. bootstrap values from , replicates are shown next to the branches. hrvc strains include strains from korea (kr), strains from wisconsin (eu and eu ), strain qpm from australia (ef ), strain - from hong kong (ef - ), strain nat from ucsf (ef ), and strain nat from ucsf (ef ). group b strains include strains from korea, hrv (ef ), and hrv (ef ). group a strains include strains from korea. nine strains from korea did not belong to groups a-c. been reported based on analysis of vp- and - genomes [ ] . lee et al. [ ] and kiang et al. [ ] reported that a genuine hrv-c, distinct from hrv-a and hrv-b, could be identified by pcr analysis based on the ncr region, and some strains that appeared to represent novel species, including the qpm strain described by mcerlean et al. [ ] , the strains by lamson et al. [ ] and hong kong strains [ ] , may be hrv-a variants rather than hrv-c. in the present study, phylogenetic analysis of the ncr region showed that qpm, hrv-c strain and hrv x were grouped into the hrv-c species, but strains could not be identified. in subsequent analysis of the vp /vp based on analysis of * bp from the vp /vp region. the phylogenetic tree was built using the neighbor-joining method with the kimura twoparameter estimation. bootstrap values from , replicates are shown next to the branches. nine strains whose groups were not determined from noncoding region belonged to hrv-a and hrv-b. the scale bar indicates the estimated number of substitutions per bases region, all of the strains that were not identified by ncr region analysis were identified as hrv-a (in cases) or hrv-b (in cases). these results indicate that the ncr may be useful for classifying novel species of hrv, but identification of serotype based on comparison of nucleotide sequences from the ncr should be used with caution. in this study, hrv-c infection did not require admission to the intensive care unit and prognosis was good, which is different from what has been found in previous studies [ , ] . in conclusion, hrv-c and hrv-a were co-circulating in children hospitalized with lrtis in korea in , implying a possible role of hrv-c in lrtis. however, further studies are needed to standardize diagnostic methods for detection of hrv-c infection and to determine its association with a severe clinical course. rhinoviruses. in: fields virology rhinoviruses infect the lower airways virological and serological analysis of rhinovirus infections during the first two years of life in a cohort of children asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection role of rhinovirus in hospitalized infants with respiratory tract infections in spain viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation virological diagnosis in community-acquired pneumonia in immunocompromised patients picornaviridae phylogenetic analysis of rhinovirus isolated collected during successive epidemic seasons clinical features and complete genome characterization of a distinct rhinovirus genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children a diverse group of previously unrecognized human rhinoviruses are common cause of respiratory illness in infants characterization of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis mass taq polymerasechain reaction detection of respiratory pathogens, including a new rhinovirus genotype, that causes influenza-like illness in new york state during novel human rhinoviruses and exacerbation of asthma in children detection of viruses identified recently in children with acute wheezing mega : molecular evolutionary genetics analysis (mega) software version . global distribution of novel rhinovirus genotype an assay for -noncoding region analysis of all human rhinoviruses prototype strains a recently identified rhinovirus genotype is associated with severe respiratory tract infection in children in germany molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality acknowledgments this study was partly supported by research grant ( ) by inje university. key: cord- -wy mtafh authors: waghmare, alpana; boeckh, michael title: rhinovirus, coronavirus, enterovirus, and bocavirus after hematopoietic cell transplantation or solid organ transplantation date: - - journal: transplant infections doi: . / - - - - _ sha: doc_id: cord_uid: wy mtafh respiratory viral infections represent a significant cause of morbidity and mortality in immunocompromised hosts. newer molecular detection assays have allowed for the characterization of several respiratory viruses not previously recognized as having significant clinical impact in the immunocompromised population. human rhinoviruses are the most common respiratory viruses detected in the upper respiratory tract of hematopoietic cell transplant and lung transplant recipients, and evidence on the impact on clinical outcomes is mounting. other respiratory viruses including enteroviruses (evs), coronaviruses (covs), and bocavirus may also contribute to pulmonary disease; however, data is limited in the immunocompromised population. further studies are needed to define the epidemiology, risk factors, and clinical outcomes of these infections; this data will help inform decisions regarding development of antiviral therapy and infection prevention strategies. human rhinoviruses (hrvs), the viruses predominantly associated with the common cold, are highly prevalent in both immunocompetent and immunocompromised individuals. prior to the development of sensitive molecular viral detection assays, infl uenza, respiratory syncytial virus, and parainfl uenza virus were the most common and most concerning respiratory viral pathogens detected in hematopoietic cell transplant (hct) recipients [ ] . due to the development of polymerase chain reaction (pcr) assays for viral detection, hrvs are now known to be the most common viruses detected from respiratory specimens in hct recipients and can account for - % of cases of viral respiratory infections in these patients [ - ] (figure - ). due to their high prevalence and their ability to cause progressive infection, hrvs are also a signifi cant cause of lower respiratory tract infection (lrti) in hct recipients (table - ) . hrv infection is also common in solid organ transplant (sot) recipients , although the incidence is not known among sot recipients as a whole. in lung transplant recipients , data from older retrospective and prospective studies suggests an incidence of - % among patients with positive respiratory samples [ - ] (figure - ) . in a recent prospective surveillance study of lung transplant recipients, hrvs represented % of all positive samples [ ] . among symptomatic lung transplant recipients, hrv represented % of all respiratory viruses detected [ ] . hrvs are members of the picornaviridae family and are classifi ed into three species, hrv-a, hrv-b, and hrv-c, based on similarity in genome organization, capsid features, and conserved sequences [ ] . the total number of genotypes continues to grow as new genotypes are characterized; currently at least unique genotypes are described. due to poor growth in traditional viral culture models, hrv-c was only recognized after the development of molecular diagnostic techniques. thus, hrv-c is not a novel species, but rather one that has been circulating unnoticed due to lack of an appropriate diagnostic assay. there are several biologic characteristics of hrv-c that differentiate the species from hrv-a and hrv-b. hrv-a and hrv-b both use icam- or ldlr for cell attachment and entry, whereas it appears that hrv-c may utilize a distinct receptor, cadherin-related family member , that is associated with asthma susceptibility [ , ] . additionally, hrv-c species are stable at higher temperatures and readily infect upper and lower airways, whereas hrv-a and hrv-b species tend to be more limited to the sinuses and upper airways [ , ] . these biologic characteristics are thought to play a role in variations in clinical outcomes observed among the different species. most immunocompetent patients with hrv present with an afebrile, self-limited syndrome characterized by rhinorrhea, nasal congestion, and malaise, and less frequently sore throat, mild cough, and hoarseness [ - ] . hrv may also be associated with exacerbations of sinusitis, chronic bronchitis, and asthma, and with lower respiratory tract syndromes and atypical pneumonias in otherwise healthy people, including the young and the elderly [ , ] . the specifi c mechanisms by which hrvs produce lung diseases are not well understood. hrvs are also implicated in asthma and chronic obstructive pulmonary disease (copd) exacerbations, but again the mechanisms are poorly defi ned. with the widespread availability of pcr diagnostics, data are emerging on the incidence and clinical relevance of hrv infections in immunocompromised patients. early studies relied on culture to detect hrv, a specifi c but insensitive method because the standard viral culture systems are not optimized for hrv detection, especially hrv-c [ ] . for example, a fred hutchinson cancer center surveillance study from to detected hrvs in specimens, and only one was from a lower respiratory tract specimen [ ] . a prospective -year study at md anderson cancer center cultured specimens specifi cally for hrvs at lower temperatures with roller culture methods, and reported that hrv infections were associated with substantial morbidity and mortality in of ( %) myelosuppressed patients [ ] . in that study, approximately one third of the adult hct recipients who developed symptomatic hrv infections prior to engraftment had progression of upper respiratory tract symptoms to lrti, and all cases with pneumonias were fatal. lung biopsies and autopsies revealed fi ndings consistent with interstitial pneumonitis and/or ards, but no in situ evaluation was performed to defi nitively assess hrv infection. similar reports with evidence of lrti based on radiographic and bal fi ndings continue to be noted [ - ] , but it remains unknown if pneumonia is a direct cause of viral invasion of the lung tissue or by host responses in the lung. evidence for in vitro and in vivo replication in lower respiratory tract has been shown in experimental infection, where hrv was isolated from human volunteers after intranasal hrv challenge by in situ hybridization [ ] . the use of rt-pcr continues to provide new information about the frequency of hrv infection. in a study of bal samples from hct recipients that were tested using rt-pcr , hrv was detected in six patients ( %), mortality rate was very high ( %) and two of the six patients showed persistent hrv infection. however, all of the hrv-infected patients had signifi cant coinfections and it was not certain whether hrv infection was the direct cause of poor prognosis [ ] . in a small cohort of patients with hematologic malignancy, lrti was associated with hypoalbuminemia and bacterial copathogens were seen in % of patients [ ] . recent studies have shown that immunocompromised adults with hrv demonstrated similar hospital admission rates, intensive care unit admissions, and mortality rates as patients with pandemic h n infl uenza [ ] . several reports have linked hrv infection to severe respiratory failure and even death [ - ] . furthermore, recently presented data suggest that lrti associated with hrv leads to a mortality rate comparable to that of rsv, infl uenza virus, and piv [ ] , independent of the presence of co-pathogens. risk factors for mortality following hrv lrti included bone marrow stem cell source, oxygen requirement at time of diagnosis, and steroid use ≥ mg/kg prior to diagnosis [ ] . other factors that may infl uence clinical severity include the presence of hrv rna in blood, viral load, and hrv species type; however, no data exist in immunocompromised patients to date. hrv viral rna was detected in the sera of ( %) of pediatric patients with severe hrv respiratory infection, with hrv-c being the predominant species [ ] . in healthy pediatric patients, increased respiratory viral load has been associated with hrv lrti and hrvc has been implicated as a more virulent pathogen [ - ] . others, however, have shown lack of correlation between hrv-c and oxygen requirement, length of hospitalizations, and coinfections [ ] . the predominance of hrv-c in hct recipients has also been described in small studies, with higher rates of pneumonia in patients with hrv-c detected from the upper respiratory tract [ ] . in a small cohort of patients with hematologic malignancies, the rate of lrti was not different between patients infected with hrv-a, hrv-b, or hrv-c [ ] . the relative risk of hrv-c infection in the immunocompromised population remains unknown, and more research is needed to defi ne the role of strain differences on outcomes. detection and diagnosis of respiratory viral infections prior to transplant is a common clinical concern that has until recently only been evaluated in small cohorts for certain viruses [ - ] . in a large, prospective surveillance cohort of allogeneic hct recipients, detection of hrv pretransplant was associated with signifi cantly fewer days alive and out of the hospital, and signifi cantly higher mortality at days posttransplant [ ] . further, larger prospective studies are needed to determine risk factors for posttransplant complications, the role of viral load and symptom burden at the time of transplantation, and the need to potentially delay transplantation for patients with hrv present prior to transplantation. ultimately, the issue of viral causality of disease and evaluation of prophylactic and treatment modalities will need to be addressed. the impact of hrv infection prior to sot is not known. like hct recipients, sot recipients are exposed to highly immunosuppressive regimens that leave them susceptible to respiratory viral infections. lung transplant recipients have the added disadvantage of altered lung immunity due to factors such as impaired ciliary clearance, poor cough refl ex, and abnormal lymphatic drainage. these factors can predispose to lower respiratory tract infections. the impact of hrv on outcomes in lung transplant recipients can range from asymptomatic infection to severe disease. in a pooled analysis of all respiratory viruses detected in lung transplant recipients, viruses were detected fi ve times more frequently when respiratory symptoms were present [ ] . a correlation between higher symptom scores and higher rhinovirus load in the upper respiratory tract has been demonstrated, although even asymptomatic patients can have relatively high viral loads [ ] . the relative rate of progression from upper to lower tract disease for hrv specifi cally is not known, although the effect on lung function has been evaluated in aggregate for all respiratory viruses and suggests a decline in forced expiratory volume (fev ) of − % to − % [ ] . for hrv specifi cally, the fev loss was similar to that seen in other respiratory viruses [ ] . the correlation between respiratory viral infections and acute rejection, chronic rejection, and bronchiolitis obliterans syndrome (bos) remains somewhat unclear, with several confl icting fi ndings when respiratory viruses were evaluated in aggregate [ - ] . a recent large cohort of lung transplant recipients, however, showed an independent association between respiratory viral infections ( % hrv) and chronic lung allograft dysfunction in multivariate models [ ] . this association was infl uenced by time, with more of an effect within a shorter period following respiratory infection. larger, prospective studies investing individual viruses are needed to clearly assess the impact on these outcomes. unlike paramyxoviruses, hrv infection cannot be diagnosed based on characteristic histopathologic changes or changes in cell morphology. in the past, cell culture was used to diagnose hrv infection using multiple cell lines at low temperatures of - °c, often in rolling tubes. the cell lines utilized for the detection of hrvs may detect enteroviruses; hrv isolates are distinguished from enteroviruses by their lability in acid (loss in viral titer following exposure to a ph of ). there are no commercially available antigen-detection assays or simple kits for the detection of hrv. rt-pcr has dramatically improved the ability to both detect and characterize hrvs, with current assays at least two to three times more sensitive than conventional culture methods [ ] . some pcr assays are able to distinguish between enteroviruses and hrvs instead of the acid lability assays [ ] . typing of hrvs based on pcr amplifi cation sequence variations in ′-noncoding region also has been described [ ] . new standardized methods to detect more of the over strains of hrv have now been described [ ] ; however, commercially available multiplex respiratory viral pcr panels contain primer/probe sets that can cross-react between enterovirus and hrv strains. new strains and types of hrv are being detected frequently and more diseases associated with hrv are being described using new and diverse molecular methods. there are no approved antivirals for the treatment of hrv infections. several agents have been evaluated in preclinical and clinical trials for the treatment of hrv infection in immunocompetent hosts, including capsid binding inhibitors, protease inhibitors, and rna synthesis inhibitors [ ] . none of these agents have been evaluated in immunocompromised hosts. given the high prevalence and potential severity of hrv infection in this population, there is a great need for drug development and clinical trials for the prevention and treatment of lrti. outside of transplant recipients, there is a potential need for intervention in other populations such as patients with asthma or copd to prevent disease exacerbation [ , ] . covs are a frequent cause of the common cold, but little is known about the role of covs in immunocompromised patients [ ] (table - ). human group (subtypes e and nl ) and human group (oc and hku ) covs were originally reported as causes of human respiratory illnesses. the availability of more sophisticated diagnostic tools, such as rt-pcr , has facilitated the detection of covs in normal and immunocompromised persons. these improved molecular methods of viral discovery facilitated the recent identifi cation of the novel group and human cov subtypes-nl in [ ] and hku in [ ] . a more accurate clinical epidemiology of cov infection is beginning to emerge. it is now known that all four known subtypes of cov circulate simultaneously [ ] , and that in addition to the common cold, cov is associated with upper respiratory tract infection and lrti in persons with and without underlying conditions [ , ] . in lung transplant recipients, covs appear to be the second most common respiratory viruses after picornaviruses with a detection rate of - % of positive samples [ - ] (figure - ) . in a prospective surveillance cohort of lung transplant recipients, coronaviruses were detected in % of all positive samples, again only second to picornaviruses [ ] . two additional covs associated with outbreaks are the severe acute respiratory syndrome-associated cov (sars-cov) and the recently described middle east respiratory syndrome-cov (mers-cov) . the sars outbreak originated in guangdong province in china in and was characterized by a life-threatening, atypical pneumonia and was spread by close contact with infected humans, mostly to household contacts and health care workers [ ] . sars-cov is not currently circulating in the world with the most recent human cases of infection reported in china in [ ] . mers-cov fi rst emerged in the arabian peninsula in , and since then travel-associated cases have been found in a number of countries outside the region [ ] . in adults, the fatality rate is estimated to be %; in children asymptomatic infection is common but patients with underlying medical conditions are at increased risk [ , ] . there is little data on the incidence of sars-cov and mers-cov in immunocompromised hosts, although immune suppression is considered a risk factor. sars-cov has been described in liver transplant recipients and in patients with myelodysplastic syndrome [ , ] . mers-cov has been described in patients on chronic immunosuppression and in renal transplant recipients with a broad range of clinical presentations [ , ] . other covs have been reported to cause pneumonia in children and immunocompromised patients treated for hematologic malignancies [ - ] . the role of coronavirus virus infection prior to transplantation is not known. although most cov infections result in relatively mild upper respiratory tract infection, these viruses have been associated with more severe lrti (e.g., bronchiolitis and pneumonia) in patients who are immunosuppressed, have asthma, or are premature. in one retrospective study carried out over year, cov was detected in six immunocompromised children-fi ve with acute lymphocytic leukemia and one renal transplant recipient [ ] . five patients were febrile at the time coronavirus was present, with fevers lasting - days. all patients initially presented with rhinorrhea and nasal discharge; two children had cough as a presenting symptom. chest radiographs of only one of the three children were abnormal; lrti based on decreased oxygen saturation, tachypnea, and abnormal chest radiograph was present in only one child with leukemia, who was signifi cantly neutropenic and lymphopenic at the time cov was detected. covs have been associated with lrti in hct recipients with sometimes fatal outcomes [ , , - ] . the clinical characteristics of sars-cov and mers-cov infection in hct and sot patients are not well described, and presentation can range from mild symptoms to respiratory failure and death [ - , ]. until the advent of rt-pcr , techniques for the detection of cov were limited and the reliable identifi cation of cov was problematic. early detection techniques isolated two subtypes-oc and e, originally using organ cultures of human embryonic trachea, with morphology determined using negative staining with electron microscopy [ ] . with the advent of molecular detection methods and increased interest in cov detection during the sars outbreak, new strains of covs have been discovered and new rt-pcr assays developed that facilitate further studies of these viruses. based on rt-pcr assays, four strains of non-sars covs (oc , e, nl , and hku ) appear to cocirculate during the non-summer months in temperate climates, and are associated with symptomatic disease in immunocompromised hosts [ ] . guidance on rt-pcr and serologic assays for the confi rmation on mers-cov can be found on the world health organization website [ ] . there are no approved antivirals for prophylaxis or treatment of cov infections and supportive care remains paramount in managing patients infected with coronaviruses. though several antivirals were used during the sars-cov epidemic, no clear benefi t could be established on systematic review [ ] . oral ribavirin was evaluated in retrospective studies for the treatment of mers-cov in immunocompetent individuals; decreased survival was noted in one study when compared to matched controls [ , ] , however, larger prospective studies are needed to show true effi cacy. shedding of all coronaviruses may persist for up to months, and routine infection control practices are encouraged. evs are part of the picornaviridae family of viruses and can be associated with severe illness in immunocompromised hosts. evs include polioviruses, coxsackieviruses, and echoviruses; these are now all classified into four species: enterovirus a (ev-a), ev-b, ev-c, and ev-d. risk for infection and subsequent poor outcomes appears to be heavily influenced by age, although factors such as sex and socioeconomic status play a role in the general population. ev activity can be either sporadic or epidemic, and several outbreaks have been described. evs are typically found during the summer and early autumn in temperate climates. enterovirus-d (ev-d ) was fi rst identifi ed in california in [ ] and has since been associated with several small outbreaks, both in the us and internationally, from to [ - ] . in the summer of , several hundred cases of severe respiratory illnesses in children in the united states were found to be associated with ev-d infection [ ] , and several additional clusters have been described worldwide [ - ] . evs can cause a wide spectrum of illnesses in immunocompetent individuals including asymptomatic infection, poliomyelitis, meningitis, encephalitis, cardiac disease, muscle disease, eye infections, respiratory infections, exanthems, and neonatal disease. the most frequently described manifestation in immunocompromised patients is respiratory disease, although the incidence and spectrum of disease is not known. according to one study, evs can be associated with lower respiratory tract infection and mortality; however, larger studies are needed to establish specifi c risk factors for worse outcomes [ ] . most confi rmed cases of ev-d infection have been in children, occurring primarily in patients with underlying lung disease such as asthma or a history of wheezing. ev-d was also associated with several cases of acute fl accid paralysis in children during the outbreak in the united states, although defi nitive causation has not yet been established [ , ] . the impact of ev-d infection in immunocompromised hosts is not known; however, the association between ev-d and severe illness was described in eight adult immunocompromised patients with presumptive ev-d infection including hct recipients [ ] . additionally, one recent report of adults with confi rmed ev-d infection included solid organ transplant recipients [ ] . depending on the clinical scenario, evs can be detected from a number of clinical specimens including cerebral spinal fl uid, serum, respiratory specimens, cardiac tissue, and stool. evs may be identifi ed in throat samples as well as fecal specimens and cerebrospinal fl uid. commercial multiplex pcr assays contain primer/probe sets that may cross react between rhinoviruses and enteroviruses. a specifi c ev-d rt-pcr has been developed by the cdc and has been made publically available [ ] . there are no approved antivirals approved for the treatment of evs. intravenous immunoglobulin (ivig) has been used in the treatment of neonatal enteroviral sepsis, but the effect on clinical outcomes is highly dependent on the presence of specifi c neutralizing antibodies and timing of administration [ , ] . pleconaril , an oral capsid inhibitor with activity against picornaviruses, has been evaluated in treatment of enteroviral infections including meningitis, neonatal sepsis, and respiratory infections [ - ] but is not available for treatment. other capsid binders, protease inhibitors, and polymerase inhibitors are in various stages of development, but none are currently available for treatment of enteroviral infections [ ] . no studies have shown effi cacy in immunocompromised hosts. human bocavirus (hbov) is a newly identifi ed human parvovirus that was originally identifi ed by random pcr amplifi cation/cloning technique on pooled respiratory secretions from hospitalized children with respiratory tract symptoms [ ] . this virus was named "human bocavirus," due to its relatedness to the genome organization of two other parvoviruses, bovine parvovirus and minute virus of canines, in the family parvoviridae. this virus continues to be detected in young children with a winter seasonality [ - ] . the relationship of hbov and respiratory disease in immunocompromised patients is not yet clear. preliminary evidence to date demonstrates case reports of disseminated hbov infection with involvement of the respiratory tract, blood, and stool in several patients, sometimes associated with gvhd and prolonged viral shedding in the feces [ , ] . other studies have reported little evidence linking this virus with pulmonary pathology or severe respiratory disease in hct or lung transplant recipients [ - ] . further research is necessary to link this virus with the disease in the transplant recipient. no specifi c antiviral therapy is available. respiratory viruses are a signifi cant concern following hct and sot and can be associated with substantial morbidity and mortality, even among viruses traditionally not concerned pathogenic. new, sensitive diagnostic assays allow for routine detection of rhinoviruses, enteroviruses, coronaviruses, and bocavirus, and additional data on the epidemiology, risk factors, outcomes of infection, and the impact of different viral strains are desperately needed. preliminary studies suggest that detection of these viruses prior to transplant may affect outcomes, but additional studies are needed to explore this important clinical area. furthermore, as new antivirals are being developed, it will be important to identify high-risk patients that may benefi t from treatment. finally, a better understanding of these viruses will be able to inform better infection prevention strategies that will remain the mainstay of viral control. respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience respiratory viral infections after bone marrow/peripheral stem-cell transplantation: the christie hospital experience human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study symptomatic respiratory virus infection and chronic lung allograft dysfunction analysis of the complete genome sequences of human rhinovirus the minor receptor group of human rhinovirus (hrv) includes hrv and hrv , but the presence of a lysine in the vp hi loop is not suffi cient for receptor binding cadherin-related family member , a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication clinical and molecular features of human rhinovirus c. microbes infect biological characteristics and propagation of human rhinovirus-c in differentiated sinus epithelial cells frequency and natural history of rhinovirus infections in adults during autumn natural and experimental rhinovirus infections of the lower respiratory tract fields virology respiratory viral infections in immunocompetent and immunocompromised persons clinical virology detection of rhinovirus rna in lower airway cells during experimentally induced infection community-acquired pneumonia requiring hospitalization among u.s. adults manual of clinical microbiology rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients rhinovirus as a cause of fatal lower respiratory tract infection in adult stem cell transplantation patients: a report of two cases rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients rhinoviruses infect the lower airways clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy severity of human rhinovirus infection in immunocompromised adults is similar to that of h n infl uenza human rhinovirus rna detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality. abstract presented at: asbmt tandem meeting detection of human rhinovirus c viral genome in blood among children with severe respiratory infections in the philippines molecular epidemiology of human rhinovirus infections in the pediatric emergency department association between human rhinovirus c and severity of acute asthma in children rhinovirus load and disease severity in children with lower respiratory tract infections molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in human rhinovirus c in adult haematopoietic after hematopoietic cell transplantation… stem cell transplant recipients with respiratory illness pretransplantation respiratory syncytial virus infection: impact of a strategy to delay transplantation respiratory virus infection in immunocompromised patients prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients parainfl uenza virus infection pre-and posthaematopoietic stem cell transplantation: re-infection or persistence? clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms classifi cation of respiratory tract picornavirus isolates as enteroviruses or rhinoviruses by using reverse transcription-polymerase chain reaction typing of human rhinoviruses based on sequence variations in the ′ non-coding region real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses the human rhinovirus: humanpathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery toward antiviral therapy/prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies rhinovirus and asthma: a storied history of incompatibility identifi cation of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia clinical disease in children associated with newly described coronavirus subtypes coronavirusassociated pneumonia in previously healthy children characterization of human coronavirus oc and human coronavirus nl infections among hospitalized children < years of age a review of studies on animal reservoirs of the sars coronavirus severe acute respiratory syndrome-retrospect and lessons of outbreak in china update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities estimation of mers-coronavirus reproductive number and case fatality rate for the spring saudi arabia outbreak: insights from publicly available data middle east respiratory syndrome coronavirus disease in children severe acute respiratory syndrome (sars) in a liver transplant recipient and guidelines for donor sars screening a major outbreak of severe acute respiratory syndrome in hong kong clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission mers cov infection in two renal transplant recipients: case report fatal lower respiratory tract disease with human corona virus nl in an adult haematopoietic cell transplant recipient coronavirus e-related pneumonia in immunocompromised patients human coronavirus oc pneumonia in a pediatric cancer patient with down syndrome and acute lymphoblastic leukemia genetic variability of human coronavirus oc -, e-, and nl -like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients coronavirus pneumonia following autologous bone marrow transplantation for breast cancer use of a novel virus detection assay to identify coronavirus hku in the lungs of a hematopoietic stem cell transplant recipient with fatal pneumonia short-term outcome of critically ill patients with severe acute respiratory syndrome viral infections of humans world health organization. laboratory testing for middle east respiratory syndrome coronavirus sars: systematic review of treatment effects ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study a probable new human picornavirus associated with respiratory diseases clusters of acute respiratory illness associated with human enterovirus -asia enterovirus among children with severe acute respiratory infection, the philippines detection of enterovirus in serum from pediatric patients with pneumonia and their clinical outcomes. infl uenza other respi viruses enterovirus in children with acute respiratory tract infections circulating viruses associated with severe complicated enterovirus infection in taiwan: a multi-year analysis a fatal central nervous system enterovirus infection severe respiratory illness associated with enterovirus d -missouri and illinois high frequency of enterovirus d in children hospitalised with respiratory illness in norway, autumn . infl uenza other respi viruses the emergence of enterovirus d in a dutch university medical center and the necessity for routinely screening for respiratory viruses molecular epidemiology of enterovirus d from to in philippines characterization of enterovirus activity, including that of enterovirus d , in pediatric patients in alberta, canada, in genome sequence of enterovirus d and clinical disease two cases of acute severe fl accid myelitis associated with enterovirus d infection in children enterovirus d infection low-level circulation of enterovirus d -associated acute respiratory infections emergence of enterovirus d in denmark clinical characteristics and molecular epidemiology of enterovirus infection in infants < months in a referral paediatric hospital of barcelona enterovirus d nosocomial outbreak in elderly people upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies a novel outbreak enterovirus d strain associated with acute fl accid myelitis cases in the usa ( - ): a retrospective cohort study a cluster of acute fl accid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus d in children in colorado clinical disease due to enterovirus d in adult hematologic malignancy patients and hematopoietic cell transplant recipients ev-d ) outbreak strain-specifi c real-time reverse transcription/polymerase chain reaction (rrt-pcr) assay instructions neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin effect of intravenous immunoglobulin for neonates with severe enteroviral infections with emphasis on the timing of administration enteroviral meningitis: natural history and outcome of pleconaril therapy oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: effi cacy and tolerability in phase ii clinical trials effi cacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses a randomized, double-blind, placebo-controlled trial of pleconaril for the treatment of neonates with enterovirus sepsis replication and inhibitors of enteroviruses and parechoviruses from the cover: cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus primary infection and shedding in infants disseminated bocavirus infection after stem cell transplant persistence of human bocavirus dna in immunocompromised children rare and emerging viral infection in the transplant population human bocavirus: passenger or pathogen in acute respiratory tract infections? absence of human bocavirus in bronchoalveolar lavage fl uid of lung transplant patients key: cord- -pbm vs h authors: trigg, c. j.; nicholson, k. g.; wang, j. h.; ireland, d. c.; jordan, s.; duddle, j. m.; hamilton, s.; davies, r. j. title: bronchial inflammation and the common cold: a comparison of atopic and non‐atopic individuals date: - - journal: clin exp allergy doi: . /j. - . .tb .x sha: doc_id: cord_uid: pbm vs h background cold virus infections are associated with asthma attacks and with increased bronchial responsiveness even in normal subjects. possible mechanisms include epithelial damage, interaction with adhesion molecules or with t‐helper cell subsets. objective to determine whether colds increase lower airway inflammation, comparing atopic with non‐atopic normal subjects. methods thirty healthy volunteers ( atopic) took part. basehne tests included viral serology. microbiological culture and polymerase chain reaction for rhinovirus infection (hrv‐pcr), histamine bronchial provocation and bronchoscopy. twenty subjects (eight atopic) underwent repeat tests when they developed a cold. results forced expiratory volume in one second (fev( )) was significantly lower during colds (‐ . l [ % confidence mterval ‐ . , ‐ . ], p= , ) and there was a significant increase in bronchial responsiveness (+ . doublings of the dose‐response slope [+ . , + . ], p= . ). eight subjects (two atopic) had a diagnosed viral infection: two hrv. three coronavirus (hcv), one hrv + hcv, one parainfluenza iii(pi) and one respiratory syncytial virus (rsv) (also haemophilus influenzae). in biopsies, during colds, total eosinophils (eg (+)) increased significantly (geometric mean . ‐fold [ . , . ], p=o. ). activated eosinophils (eg (+)) only increased significantly in the subgroup without diagnosed viral infection and particularly in atopic rhinitics. t‐suppressor (cd (+)) cells also increased significantly (median + . cells mm( ), p= . ). epithelial expression of intercellular adhesion molecule‐ (icam‐ ) expression increased in four atopic rhinitics during colds. bronchial washings showed a significant increase in neutrophils (gm . ‐fold [ . , . ], p= . ). conclusion lower airway inflammation was present in atopic and non‐atopic normal subjects with colds. atopic subjects differed in that they were less likely to have positive virological tests and were more likely to show activated eosinophilia in the lower airway, despite a similar spectrum of symptoms. attacks with upper respiratory tract viral infections, a majority of which are rhinoviral [ ] [ ] [ ] [ ] [ ] . until recently. there is increasing evidence of an association of asthma rhinovirus (hrv) infections were difficult to diagnose genome common to many if not all serotypes, allowing improved diagnosis of hrv infection. two studies using this technique have found particularly high identification rates for hrv in asthma exacerbations [ . ] . several studies have shown that colds may increase asthma severity, using indirect measurements, such as peak expiratory fiow (pefr), bronchial responsiveness and the late asthmatic response to allergen [ ] [ ] [ ] . however, there is little information on the direct effect of cold virus infection on the immunopathology of the lower respiratory tract. bronchial biopsies in influenza and mycoplasma have shown inflammatory features consistent with asthma [ , ] . nasal biopsies in experimental rhinovirus infection have not shown evidence of an inflammatory infiltrate [ ] and it is suggested that mediators such as histamine and bradykinin may cause upper airway symptoms [ ] . in nasal epithelial cell cultures, rhinoviruses have a negligible cytopathological effect, compared with the destruction of the monolayer caused by influenza a and adenovirus [ ] . nevertheless, nasal epithelial cell shedding is increased in vivo in experimental rhinovirus infection [ ] . given the high rates of rhinovirus infection in association with asthma exacerbations, it is intriguing to note that the receptor for major rhinovirus serotypes is the intercellular adhesion molecule, icam- [ ] . its ligands include integrins present on inflammatory cells and binding to icam- is essential to migration of inflammatory cells into tissues [ , ] . icam- is constitutively expressed by vascular endothelial cells but is also upregulated on bronchial epithelial cells in asthma [ , ] , suggesting a mechanism for inflammatory infiltration of the epithelium. viral infection may upregulate tcam- : by a direct efl'ect as shown in cultured tumour cell fines [ ] or indirectly via expansion of th| t-helper cell clones producing interferon-- [ . ] . the aim of this study was to examine bronchial epithelial tissue in naturally acquired cold virus infections, in order to determine whether there are differences between the lower airway infiammatory response and icam- expression in atopic and nonatopic subjects. we decided to study naturally acquired colds, using respiratory symptoms as the diagnostic endpoint, as experimental infection with a specific rhinovirus serotype may not necessarily result in the spectrum of symptoms and inflammatory changes which occur in common colds. we anticipated ^ % identification of viral infections, with a predominance of rhinovirus infections as in previous studies [ . ] . asthmatics were not studied as we felt that it would be inappropriate to bronchoscope such subjects during an exacerbation. atopic subjects show mild bronchial infiammatory changes [ ] which may be exacerbated by environmental factors, without unacceptable risk. thirty normal individuals aged between and years gave written informed consent to take part in the study, which was approved by the district research ethics committee. none of the subjects had a history of asthma nor chronic cough, wheeze or dyspnoea. subjects were excluded if they had smoked in the preceding years or if they had a past history of more than packyears of smoking. fifteen subjects were selected for atopy, on the basis of one or more positive skin-prick tests to a screen of five common inhalant allergens. all had normal spirometry forced expiratory volume in one second (fevi) greater than % predicted for age and height [ ] . subjects were recruited between november and march at a time when they had not had a cold for at least weeks. screening investigations included a brief respiratory symptom questionnaire, medical examination, skin-prick tests to five common inhalant allergens, simple spirometry, histamine bronchial provocation test and a -day diary card of respiratory symptoms and peak expiratory flow. subjects were reviewed after days and excluded from the study if their diary cards revealed greater than % diurnal peak fiow variability or chronic lower respiratory symptoms. blood was taken for platelet count, clotting screen and viral serological tests. nose and throat swabs were taken for hrv-pcr. bronchoscopy was performed - days later. bronchial washings were taken for microbiological culture and hrv-pcr and bronchial biopsies for immunopathological studies. subjects were reviewed regularly at monthly intervals and asked to attend for review at the onset ofa cold with lower respiratory symptoms (cough, wheeze or dyspnoea). diary card symptom scores were reviewed in order to confirm the diagnosis of a cold. diagnosis was based on the recording of cough together with at least one other symptom (nasal blockage, rhinorrhoea, wheeze or dyspnoea), provided that at least one symptom was rated grade (moderate) or more [ ] . symptom scores were confirmed at interview with the doctor. clinical examination was performed in order to exclude serious respiratory disease. histamine bronchial provocation test, polymerase chain reaction (pcr) on nose and throat swabs and viral serology were repeated, followed by bronchoscopy - days later, when bronchial washings and biopsies were taken and processed as before. bronchial biopsy sites were randomized so that g) blackwell science ltd. clinical and experimental allergy. , - half of the subjects underwent biopsy from the right tipper and half the right lower lobe at basehne, the alternate site being biopsied during the acute cold. diary cards of respiratory symptoms and peak fiow were recorded for weeks, commencing at the onset of cold symptoms. convalescent viral serology was repeated at the end of the month. questionnaire and diary cards subjects completed a written questionnaire on respiratory symptoms. subjects were excluded if they reported wheeze or breathlessness, in the absence ofa cold, at any time during the preceding months. diary cards were completed for a week at baseline, recording symptoms of cough, wheeze, breathlessness, nasal blockage and runny nose, twice daily. symptoms were graded - (nil, mild, moderate, severe). peak expiratory flow was recorded on rising and retiring to sleep, using a mini-wright meter. subjects were instructed to record the best of three reproducible readings. individuals with > % diurnal peak flow variation and those with chronic cough, wheeze or dyspnoea were excluded. diary cards were recommenced at the onset of a cold and recordings of peak flow and symptoms continued for i month. spirometry and bronchial provocation testing fevi was recorded after resting for - mln as the best of three repeatable measurements using a vitalograph dry bellows spirometer (vitalograph ltd, buckingham, uk). histamine bronchial provocation testing was performed according to a standard tidal breathing method [ ] , provided that resting fevi was greater than % predicted value for age and height [ ] , a devilbiss nebulizer driven by air at a how rate of lmin ' (output . nil in min) was used. challenge was terminated when the fev, had fallen by % compared with the lowest value after control solution (coca's solution) or at the maximum concentration ( mg/ml"' histamine). dose-response slope (sl) [ ] was calculated as the maximum percentage fall in fev, divided by the cumulative dose of histamine delivered. this parameter was used in place of provocation concentration as many subjects were only minimally responsive to histamine, and did not bronchoconstrict by % at the maximum dose. skin-prick tests were performed using skin-testing solutions (alk) dermatophagoides pteronyssinus, mixed grass pollen. aspergillus fumigatus, cat fur, dog hair, plus positive and negative controls (histamine mg/ ml"' and . % sahne). subjects were classified as atopic on the basis of one or more positive skin-prick tests, defined by a skin weal at least mm in diameter and mm greater in diameter than the negative control. bronchoscopy subjects attended for bronchoscopy at s. h. having fasted from midnight. following intramuscular pre-medication ( - mg pethidine, . mg prochlopererazine. . mg atropine), % lignocaine solution was applied to the fauces, % lignocaine solution to the vocal cords and % to airways. an olympus bf bronchoscope was passed and bronchial washing taken from the right middle lobe, using two ml aliquots of normal saline. cup forceps were used to take biopsies from right upper or lower lobe scgmental/subsegmcntal orifices, according to the randomization sequence. virological and microbiological investigations nasal swabs were taken from the anterior nares and throat swabs passed firmly over the pharynx and tonsils. swabs were placed into . ml transport medium containing nutrient broth ( % fetal calf serum, penicillin, streptomycin and amphotericin b). swabs and . ml aliquots of bronchial washings were stored at - c. specimens were subsequently analysed using a seminested reverse transcriptase (rt) pcr for human rhinoviruses (hrv) that incorporated a touchdown reaction cycle, as previously described by ireland et al. [ ] . the primers and probes used had the following sequences: rt lopcr'cggacacccaaagtag ' pcr + 'gcacttctgtttcccc ' hrv opcr- 'ggcagccacgcaggct ' pcr + 'gcacttctgtttcccc ' cultured human rhinoviruses of several serotypes were used as positive controls and virus transport medium was used as negative control for each assay. the appearance of a base pair amplification product was taken to indicate hrv infection. serum samples (loml) were taken at baseline and in the acute and convalescent phase of subsequent infection and stored at - o'c. sera were tested for complement fixing antibodies to adenovirus, influenza a and b, respiratory syncytial virus (rsv), parainfiuenza viruses - , mycoplasma pncumoniac and chlamydia psiitaci. an enzyme-linked immunosorbent assay (elisa) was used to detect rises in antibody levels to coronaviruses e and oc . sera were tested at a dilution of : ioo and a consistent ratio of > . between absorbance values of convalescent and acute samples was taken as indicating recent infection. bronchial washings were sent at baseline and in the acute phase of infection for standard microscopy and culture for bacterial pathogens. virological cultures were not there were no significant differences between atopic and non-atopic subjects. performed as our co-workers [ ] previously found no improvement in diagnostic yield when culture procedures were added to serology and hrv-pcr. a study of children [ ] showed a higher rate of picornavirus identifications in asthma exacerbations but the rate of confirmed hrv infection was similar. the excess of picornaviruses may have been unconfirmed hrvs or other enteroviruses. a higher prevalence of hrv infection may be explained by the different age group studied. all samples were processed and analysed without knowledge of subject, viral infection status or symptoms, in order to avoid bias in the interpretation of results. one biopsy was fixed in carnoy's solution, processed in a shandon citadel automatic tissue processor (shandon southern products ltd, runcorn. uk). embedded in paraffin wax and cut to //:m sections. sections were then stained with monoclonal antibodies egl (total eosinophils) and eg (activated eosinophils) (pharmacia. milton keynes, uk) using a streptavidin immunoperoxidase method. two biopsies were embedded in optimum cutting compound (agar, stanstead, uk), snap-frozen in liquid nitrogen and stored at - o'c. cryostat sections were cut at //m and stained using monoclonal antibodies for cd (total). cd (helper). cds (suppressor) t cells and cd (interleukin- receptor positive cells) (dako ltd, high wyeombe. uk) using the avidin biotin complex (abc) technique. total cell counts were made on each biopsy section and expressed per mm"^ area of tissue. areas were measured using computerized image analysis (kompira ltd, strathclyde, uk). sections were also stained for the intercellular adhesion molecule, icam- (cd ) (serotec, oxford, uk) using the abc technique. epithelial expression of icam- was graded on a scale of - by colour intensity and distribution of staining. specimens were processed immediately on lavage fluid chilled to ''c. aliquots ( //l) were cytocentrifuged at rpm for min. specimens were air dried, then stained with diffquick reagents for differentia! counts. a diffferential cell count was made by counting cells in consecutive high power fields. data were tested for a normal distribution (normal probability plot and the shapiro-wilk test) and logtransformed if appropriate. parametric tests were applied to pefr, fev], log-transformed histamine dose-response slope and log-transformed eosinophil and neutrophil data. lung function and histamine challenge data during colds were compared with the mean baseline value, using a paired r-test. paired /-tests were used to compare baseline and acute phase eosinophil and neutrophil data. two sample /-tests were used to compare changes in eosinophils. neutrophils and bronchial responsiveness during colds, between atopic and nonatopic subjects, between viral diagnostic groups and between symptom groups. for non-normally distributed data (t cells, cd -i-cells). wilcoxon's signed rank test was used to compare baseline and acute phase results and the mann-whitney {/-test to compare changes in t-cell infiltration during colds between atopic and non-atopic subjects, between viral diagnostic groups and between symptom groups. fevi. forced expiratory volume in one second. sl. slope of the bronchia! provocation dose response curve. % cl yo conftdence interval of the mean. where the confidence interval includes zero, the difference is non-significant at the % level. * significant change within group: p < - (paired /-test). ** significant change within group: p < (paired /-test). thirty subjects, mean age years (range - years) took part (table ) . sixteen were male and female. fifteen were atopic, of whom eight were rhinities, six being grass pollen-sensitive and two house dust mite sensitive. mean fev| was vo predicted (range - vo predicted) and mean pefr variation (amplitude as a percentage of mean) was . % (range - . - . %). three subjeets had bronchial hyperresponsiveness, reacting with > % fall in ffv, to concentrations of histamine lower than mg/ml '. there were no significant differences in age. sex ratios, lung function nor bronchial responsiveness between atopic and nonatopic groups. twenty subjects returned with symptoms of a cold between november and august . none ofthe pollen-sensitive rhinitics presented with colds during the pollen season. two subjects defaulted from follow-up. eight other subjects did not experience symptoms of a cold during foilow-up. viral identifications were made in eight ( %) ofthe symptomatic subjects. there were two rhinovirus (hrv) infections, three coronavirus (hcv) infections (two of serotype oc and one e), one with dual infection (pcr positive for hrv and serology positive for hcv e), one parainfluenza iii and one respiratory syncytial virus (rsv) infection. none ofthe subjects had positive hrv-pcr at baseline nor high titres on viral serology. a bacterial pathogen was identified in only one case: haemophilus influenzae was isolated from the bronchial washings in the subject who also had rising rsv titres. in addition to considering the efiect of atopy., we also considered the effect of positive virological diagnosis on symptoms, lung function, bronchial responsiveness and immunohistology. the aim of this analysis was to determine whether there were any gross differences between subjects with and without positive virological tests which could be accounted for by infection, albeit by a heterogeneous group of pathogens. although there tnay be differences between the different viral infections, numbers of subjects with any specific infection such as hrv were too few to consider as a separate subgroup in this study. all subjects complained of cough and rhinorrhoea. nine subjects complained of wheezing, five of whom had positive virological tests. nine subjects complained of breathlessness, five of these having positive virological tests. there were no significant differences in symptom severity between those with and without positive virologica! tests. eight atopic and non-atopic subjects experienced cold symptoms. only two of the eight atopic subjects had positive virological tests compared with six ofthe non-atopic subjects. only two atopic subjects complained of wheeze, compared with seven non-atopic subjects. only one atopic subject complained of breathlessness, compared with eight non-atopic subjects. symptom severity was significantly greater for breathlessness in the non-atopic group (median score compared with ., mann-whitney (;-test, fev i was significantly lower during colds with a mean reduction of . l( % ci - . , - . l, p= . ) ( table ). pefr and peak flow variation showed no significant change. there was a small but significant increase in bronchial responsiveness, shown by a mean increase of . doublings of the slope (ds) of the dose response curve ( % ci + . ,+ i. ds, /»- . ). table shows the mean changes in fev, and sl in atopic and non-atopic subjects and in those with and egl and eg -total and activated eosinophils respectively. cd , cd and cd -total, helper and suppressor t cells, respectively. il- rinterleukin- receptor positive cells. there were no significant difierences between the numbers of inflammatory cells present in the atopic and non-atopic subjects' bronchial rnucosa at baseline. numbers of t-suppressor (cd "^) eells were greater in the atopic subjects (* borderline significance; p = , mann-whitney-f/ test). without positive viral diagnoses. there was a greater increase in bronchial responsiveness in non-atopic subjects and those without diagnosed viral infection but the difference between groups was not significant. comparison of infiammatory cell infiltration of the bronchial mucosa in aiopic and non-atopic subjects at baseline (table ) there were no significant differences between atopic and non-atopic subjects when ceil counts were compared at baseline. however, there was a greater number of t cells in the atopic group which approached the % level of significatice (mann-whitney f/-test. p^ . ) only for cd positive t cells. there was a wide scatter of inflammatory cell numbers between subjects some of which may be accounted for by technical factors such as sampling and processing and also by pathophysiological factors. the scatter of results was no more marked than in other studies in the literature. eosinophil staining is shown in fig. i sixteen subjects had satisfactory bronchial biopsy samples for eg staining at each bronchoscopy. seven of these showed an increase in activated (eg ) eosinophil infiltration during colds. there was no significant increase in eg infiltration during colds. tceil immunostaining is shown in fig. . fourteen subjects had satisfactory bronchial biopsy samples for t-ccll staining. one ot these did not have a satisfactory section for total t-cell staining (cd ). t-cell numbers were not normally distributed and non-parametric statistics were used to analyse the data. there was a significant increase in cd (t-suppressor) cells from a median of . cells mm "^- . ceils mm " (wilcoxon's signed rank test, p - . ). there was also an increase in il- r positive cells, although numbers were very small (increase from median of . cells mm "- . cells mm ". p = . ). figure shows the differential cell counts in bronchial washings. seventeen subjects had satisfactory bronchial cytology specimens for differential cell count. there were no significant changes in macrophages, lymphocytes, eosinophils or epithelial cells. however, there was a significant increase in the percentage of neutrophils, from a geometric mean of . % to . %. (paired /-test: geometric mean . -fold increase, confidence interval . . -fold, p = . .) the effect of atopy and viral diagnosis on changes in infiammatory cell infiltration of the bronchial mucosa is shown in table . the table shows the change from baseline in numbers of inflammatory cells in atopic and non-atopic subjects and in those with and without positive viral diagnoses. there was no difference in change in egl infiltration between atopic and non-atopic subjects, nor between those with and without diagnosed viral infection. however, there were diflerences between changes in eg infiltration in the subgroups (graphically represented in fig. ). only two of the eight subjects with diagnosed viral infection showed an increase in eg infiltration compared with five of the eight subject without diagnosed infection. those without diagnosed viral infection showed a significant increase in eg infihration (paired /-test, p = . ). the only subject with diagnosed infection to demonstrate a large increase in eg infiltration was the subject who had dual infection with rsv and haemophilus influenzae. this subject was also an atopic (grass pollen allergic) rhinitic. there was a significant increase in eg cells in those without diagnosed infection compared with those with diagnosed infection (two sample /-testing, p - . ) atopic subjects were more likely to show an increase in eg infiltration than non-atopic subjects. the difference between changes from baseline in the atopic and non-atopic subjects was significant on two sample /-testing (p = . ) as there was a reduction in eg numbers in a majority of the nonatopic subjects. three of the four atopic subjects who showed an increase in eg eosinophils were rhinitics. there were no significant differences between atopic and non-atopic groups nor between those with and without diagnosed viral infection for t cells. there was a greater increase in neutrophils in bronchial washings in those with diagnosed viral infection but this increase was not significantly difterent from the change in those without diagnosed infection. table illustrates the association between lower respiratory tract symptoms and bronchial infiammatory cell infiltration. the table shows fig. . neutrophil count in bronchial lavage specimens. these data were log-normally distributed. statistical analysis as in pig. . the majority of samples ( / ) showed an increase in neutrophilia during acute coryzal symptoms. although the mean increase was small, it was statistically significant. table . -fold changes with confidence intervals (cl) which do not incltide ihe value . (no change) indicate a significant increase or decrease in numbers of cells. * significant change from baseline within tirotip (p < - ). paired /-test, (wilcoxon signed rank test for t cells). ^ borderline significant change from baseline within group {p =- ' ). wilcoxon signed rank test. * significant dilterence between atopic, non-atopic or viral diagnosis groups {p < ). comparison of changes: two sample /-test. in the groups of subjects who did or did not report symptoms of wheeze and dyspnoea during their colds. subjects who wheezed during their colds showed an increase in egl. cd and l- r ' cells in brotichial mucosa but this was not significantly different in comparison with the change iti those who did not wheeze. there were no significant differences between symptom subgroups for eg , cd * and cd "^ positive cells in bronchial tnucosa nor for neutrophils in bronchial washitigs. subjects with dyspnoea during their colds showed a significant increase in cds ' cells in comparison with the non-dyspnoeic subgroup (median increase + . cells mm -compared with + . cellsmm ". mann-whitney tz-test p^ . ). these subjects also showed an increase in il- r^ cells which did not differ significantly frotn the non-dyspnoeic subgroup. there was no significant association of dyspnoea with changes in other infiamtnatory cells, although there was an increase of neutrophils in bronchial washings in the non-dyspnoeic subgroup. thirteen subjects had satisfactory bronchial biopsy samples for icam- staining. icam-l staining of the epithehum was only found in four of seven atopic subjects and none of the six non-atopic subjects. icam-l expression was upregulated during colds in all of the four atopic subjects: on a grading scale of - . two subjects increased expression from i to . one from to and one from to . only one of these four subjects had an identified viral infection, this being the subject with rsv infection together with haemophilus influerizae (grade ). all the atopie subjects for whom biopsy tissue was available were rhinities. sections were available for five of the eight subjects with viral infection (one hrv. two hcv. one hrv/hcv, one rsv/ /. injlucuzac]. this study was designed in order to determine the effect of the common cold on lower airway inflarnmation. comparing the response in atopie and non-atopic subjects. the study has shown the presence of bronchial inflammation in healthy adult subjects complaining of symptoms of the comtnon cold. we demonstrated two distinct groups among such subjects: those with virologically proven infections and those without. a relative rise in neutrophils was a characteristic of the former group, whereas activated eosinophilic infiltration was present in the latter. despite such differences, symptom scores were similar in the two groups. atopy and rhinitis were factors associated with the development of activated eosinophilic inflammation in the bronchial mucosa. the majority of subjects showed reduced fevi and increases in airways responsiveness, neutrophils. total egl ^ eosinophils and cd ^ suppressor t-lymphocytes. regardless of atopy or the ptesence of viral infection. while perception of symptoms of the common cold by subject or observer eould have influenced the results of lung function and bronchial provocation testing, staining and counting of biopsy and iavage specimens was performed without knowledge of subject, eoryzal symptoms or infection status. our ilndings indicate that many subjects with symptotns traditionally thought to indicate upper respiratory tract infection have evidence of lower respiratory tract inflammation. it seems likely that total eosinophils and neutrophils increased as part of an acute granulocyte infiltrate, whereas cd ' t-suppressor cells may be involved in a killer cell response to viral infection: their role in those subjects in whom we were unable to identify a pathogen is less clear. numbers of subjects with hrv infection were too few to consider separately. however, our methodology for hrv rt-pcr has previously been well validated [ ] , results in adult asthtna exacerbations comparing well with those of a study in children using similar techniques [ ] . in the latter study there was a higher rate of picornaviruses identified by pcr but the confirmed rate of hrv was similar. the excess of picornaviruses may have been accounted for by hrv or enterovirus infections, the former being more likely. differences between the two studies may reflect the age groups involved. bronchial immunopathology in the hrv cases was not distinctive, despite lcam- being the receptor for major hrv serotypes [ ] . icam- expression in bronchial epithelium was limited to four atopic rhinilic subjects. we have not previously been able to show bronchial epithelial icam- expression in non-asthmatics, whereas icam- is constitutively expressed by the vascular endothelium. however, our previous study compared asthmatics with non-atopic non-rhinitics [ ] , thus, the findings of this study suggest that icam- may be up-regulated in lower airway epithelium in allergic rhinitis as well as in asthma. icam-i was only expressed in diagnosed viral infection in one atopic rhinitic subject with the combination of rsv together with haemophilus influenzae infection. this was a -year-old heterosexual who had never smoked. he had no exceptional symptoms and made a full and rapid recovery. bronchial lavage demonstrated high neutrophilia ( %). although this is a small study, we believe that our findings suggest a tendency, often suspected in clinical practice, for allergic subjects to report allergic inflammation as a "cold". this could explain the low diagnostic rate of viral infection particular to the atopic group, despite a combination of pcr and serological diagnostic techniques which have given a high yield of viral diagnoses in previous studies [ ] , it is interesting that there were no significant difl'erences in symptoms among atopic and non-atopic subjects save for breathlessness. which was, paradoxically, more common among the non-atopic subjects. lower respiratory tract symptoms were quite common among these subjects with uncomplicated colds, providing a clinical correlation with the inflammatory infiltrate, especially with cd '^ t cells. other investigators have found slight differences in symptomatology in atopic subjects with experimental hrv infections: symptoms tending to be earlier in onset [ ] and more severe in those with evidence of preceding hrv infection [ ] , other studies have shown that markers of allergic inflammation can be detected at distant sites in atopic subjects. djukanovic et al. [ ] showed greater numbers of inflammatory cells in the bronchial mucosa of atopic rhinitics than in non-atopic normals. in contrast, we found no difference between atopic and non-atopic subjects at baseline, but only half of our atopic subjects had rhinitis. other studies from our laboratory have shown constitutive up-regulation of cytokines such as granulocyte macrophage-colony stimulating factor (gm-csf) in the nasal mucosa of atopic non-rhinitics with eczema [ ] . thus, it is perhaps not surprising that activated eosinophils can increase in the bronchial mucosa in atopic rhinitics with episodes of respiratory symptoms. previous studies of the immunopathology of the common cold have concentrated on experimental hrv infection, examining mainly nasal secretions and peripheral blood. there have been few studies of bronchial pathology. halperin et al. [ ] isolated hrv from the lower airway in experimental infection, using a bronchoscopic technique which minimised contamination from the upper respiratory tract. calhoun et al. [ ] found that subjects experimentally infected with hrv showed an increase in eosinophils in bronchoa[veolar lavage specimens following allergen challenge. there was an amplified response in atopic rhinitic subjects, consistent with the increase in allergen responsiveness among similar subjects in a previous study [[ ] . other studies of colds have produced conflicting data. neutrophilia has been consistently found in nasal secretions [ . ] , together with shed epithelial cells [ ] . levandowski [ ] showed a reduction in cd + and to a lesser extent, cd ^ t cells in peripheral blood after hrv infection and suggested that this might indicate recruitment of such cells to the site of inflammation. however, the same investigators found only neutrophils and monocytes in nasal secretions [ ] . others have not been able to find an increase in inflammatory cells in nasal biopsies [ ] and suggested tbat the symptoms of common cold infection might be caused by mediators such as histamine and kinins [ . ] . hsia et al. [ ] showed an increase in production of interleukin- and interferon--j by peripheral blood mononuclear cells, inversely related to severity of the cold. they also showed an increase in natural killer (nk) cell-mediated cytotoxicity and antigen-stimulated blastogenesis. skoner et al. [ ] showed increases in peripheral blood total leucocytes and lymphocytes, especially, cd , cd , cds and cd ' dr^; but a decrease in nk cell activity and lymphoproliferative response to rv , particularly in nonatopic subjects. there were subtle differences in the peripheral blood response between atopic and nonatopic subjects with a tendency to a later response in the atopic allergic group. such peripheral blood changes indicate a systemic immune response to hrv. our findings indicate that healthy non-asthmatic subjects with symptoms of the common cold can develop bronchial inflammation, including an infiltrate of eosinophils. neutrophils and cd ^ t-suppressor cells. atopic rhinitic subjects were most likely to show allergic inflammation with activated eosinophilia and were least likely to have a diagnosed viral g) blackwell science ltd, clinical and e.xperimenlal allergy. . - infection. therefore, symptoms typical of the common cold may be caused by airway inflammation in the absence of viral infection. exposure to aeroallergens and possibly environmental pollutants should be considered as alternative triggers of cold symptoms. routine antibiotics in hospital management of acule asthma the association of viral and bacterial respiratory infections with exacerbations of wheezing in young astlimalic children respiratory viral infection and wheezy bronchitis in childhood viral and bacterial infections in adults with chronic asthma virul infections as a precipitant of wheeze in children: combined home and hospital study use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms improved detection of rhinoviruses in nasal and throat swabs by semi-nested rt-pcr mechanisms of bronchial hyper reactivity in normal subjects after upper respiratory tract infection effect of atopy on the natural history of symptoms, peak expiratory flow and bronchial hyperresponsiveness in and -year-old children with cough and wheeze rhinovirus upper respiratory tract infection increases airway hyperreactivity and late asthmatic reactions bronchotracheal response in human influenza bronchial epithelium in humans recently recovering from respiratory infections caused by influenza a or mycoplasma nasal biopsies in human rhinovirus infection: an immunohistochemical sttidy kinins are generated during experimental rhinoviral colds respiratory virus infection of monolayer culttires of human nasal epithelial cells shedding of infected epithelial cells in rhinovirus colds rothlein r ct al, a cell adhesion molecule icam-i is the major surface receptor for rhinoviruses intercelliilar adhesion moiecule- (icam- ) in the pathogenesis of asthma hla-dr and icam- expression on bronchial epithelial cells in asthma and chronic bronchitis the expression of intercellular adhesion moiccule- and the ,:*l-integrins in asthma selective induction of intercellular adhesion molecule- by interferon-gamma in human airway epithelial cells peripheral blood mononuclear cell interleukin- and intcrferon-gamma production, cytotoxicity and antigenstimulated blastogenesis during experimental rhinovirus infection induction by ll-i and interferon-gamma: tissue distribution, biochemistry and function of a natural adherence molecule (icam- ) quantitication of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry changes in the normal maximal expiratory flow-volume curve with growth and aging a general practice survey of bronchial hyperresponsiveness and its relation to symptoms, sex. age, atopy and smoking analysis of dose-response curves to methacholine theory and practice of histological techniques use of avidin-biotin complex (abc) in immunoperoxidase techniques, a comparison between abc and unlabellcd (pap) techniques rhinovirus infection in allergic and nonailergic subjects amplified rhinovirus colds in atopic subjects synthesis of tnf-tt, il- and gm-csf by cultured nasal epithelial cells from atopic and non-atopic non-rhinitic subjects and the effect of exposure for h to nitrogen dioxide (n ) pathogenesis of lower respiratory tract symptoms in experimental rhinovirus infection experimental rhinovirus infection potentiates airway inflammation only in allergic subjects nasal secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection acute-phase decrease of t-lymphocyte subsets in rhinovirus infection analysis of nasal secretions during experimental rhinovirus upper respiratory infections eflfect oi' rhinovirus infection on cellular immune parameters in allergic and non-allergic subjects cjt was supported by a bursary from the joint research board of st bartholomew's hospital and a travel grant from the robert malcolm trust. dci was supported by a gram from the cystic fibrosis trust. we thank the volunteers for their co-operation, julie kent, moises calderon and ray sapsford for technical assistance and janice thomas for statistica! advice. key: cord- -qx qdjmt authors: nirwan, sonam; kakkar, rita title: rhinovirus rna polymerase: structure, function, and inhibitors date: - - journal: viral polymerases doi: . /b - - - - . - sha: doc_id: cord_uid: qx qdjmt human rhinovirus is responsible for causing % of common cold infections in infants and adults. it belongs to the picornavirus family of nonenveloped positive-strand rna viruses. the rna synthesis of rhinovirus is carried out by rna-dependent rna polymerase, also known as d(pol). it catalyzes the synthesis of negative-strand rna using a positive-strand template. the structure of the enzyme consists of three domains: palm, fingers, and thumb domains and mg( +) in the active site. these conserved structural features of the enzyme help in catalyzing phosphodiester bond formation between the two consecutive nucleotide units complimentary to the template rna using a vpg primer. owing to the presence of over serotypes of the enzyme, designing specific inhibitors targeting the polymerase is a challenging task and until now no clinically approved antirhino viral drug is reported. in this review, we have given detailed information about the structure and function of the enzyme and also discussed some of the inhibitors and their in vivo activity against d(pol). in some genera l) as well as some stable precursors necessary for virus replication in host cells (paul, ) . out of these nonstructural proteins, a key component of the replication machinery of the picornavirus family is the rna-dependent rna polymerase (rdrp), also referred to as d polymerase ( d pol ). the rdrp (also known as d pol ) belongs to a superfamily of template directed nucleic acid polymerases, including dna-dependent dna polymerases (dddps), dna-dependent rna polymerases (ddrps), and reverse transcriptases (rts). all these enzymes share a cupped right-hand structure (including fingers, palm, and thumb domains). rdrps exist in unique "closed-hand" conformation, in contrast to the "open-hand" found in other polynucleotide polymerases. this closed conformation comprises several motifs (aÀd) that are the conserved features of particular rdrps and performs different roles in the catalytic activity of the enzyme. crystal structures of three serotypes (hrv b, hrv , and hrv ) have been reported (love et al., ) . these crystal structures provide deep insights about the structural arrangement and the conformational dynamic changes associated with the enzyme. using positive-sense rna template, the enzyme synthesizes daughter minus rna strand using a primer-dependent mechanism. the primer utilized in the mechanism is the uridylylated form of vpg protein. the process of uridylyation of vpg is also performed by d pol . based on this positive-sense rna template, the correct ntp molecule is added to the À nucleotide long primer resulting in the growing double-stranded daughter strand. three well-defined channels have been identified in the rdrp structures, serving as the entry paths for template (template channel) and for nucleoside triphosphates (nucleoside triphosphate channel), respectively, and the exit path for the double-stranded rna product (central channel). it performs the function of catalyzing phosphodiester bond formation through a conserved two-metal ion mechanism, wherein the two metal ions are present in the active site of the enzyme (steitz, ) . together with other viral and host proteins, this enzyme performs its function of replicating the rv rna genome in the cytoplasm of host cells. as this class of polymerases is unique to rna viruses and has no host counterpart, the viral rdrp represents a very attractive target for antiviral design. this biochemical activity is not present in mammalian cells; thus, very selective inhibitors could be designed that target only the virus without producing side effects. however, owing to the existence of more than serotypes and variability at the antigenic site, efforts made at vaccine development are hindered. to date, specific drugs that prevent or reduce rv infection are not available. thus, this opens a wide avenue to design novel "anti-rhinoviral" drugs. hrv is classified into three genetically distinct groups: hrv-a (containing serotypes), hrv-b (containing serotypes), and hrv-c. hrv-c is a novel group discovered by the international committee on taxonomy of viruses in (lamson et al., ) . another classification is based on the nature of the receptor group utilized by the polymerase enzyme in infecting the host cell. out of distinct serotypes of rvs identified, % of these utilize icam- (intercellular adhesion molecules) as a receptor for infecting hela cells and are thus referred to as the "major" receptor group (tomassini and colonno, ; greve et al., ) . all the remaining rv serotypes, with one exception, utilize the ldl (low-density lipoprotein) receptor (hofer et al., ) and are referred to as the "minor" receptor group. a systematic evaluation of a group of capsid-binding agents ( compounds) against all serotyped hrv revealed the existence of two antiviral groups, a and b, based upon differential susceptibility to these antiviral compounds. hrv and poliovirus (pv) belong to group a, while hrv and hrvia belong to group b (andries et al., ) . the crystal structures for the three main serotypes (hrv b, hrv , and hrv ) of the enzyme were solved by love and his group. all the three crystal structures superimposed closely, but the largest difference arose in the external residues and this could arise from crystal packing as well as sequence variation (love et al., ) . hrv d pol displays the typical "right-hand" arrangement of fingers, palm, and thumb domains and its folding topology resembles that of rdrps from hepatitis c virus (hcv) (ago et al., ; lesburg et al., ) and rabbit hemorrhagic disease virus (rhdv) (ng et al., ) . also, hrv d pol resembles most closely to pv d pol enzyme (hansen et al., ) , except at the n terminus. excluding pv residues À , superposition of the two gives a root mean square deviation (rmsd) of . Å for hrv ( % identity), . Å for hrv ( % identity), and . Å for hrv b ( % identity), and that is why pv rdrp is sometimes considered while studying inhibitors for hrv rdrp. the complete view offered by hrv d pol confirms a conservation of tertiary structure among rdrps from diverse rna viruses and that is why rdrps is a very promising target for developing multitarget antiviral drugs having broad range of biological activity. differences observed in the secondary structure of these polymerases reflect not only the substrate diversity but also divergent mechanisms for initiation of rna synthesis (primer dependent for hrv and rhdv but primer independent for hcv and bacteriophage ϕ ). three well-defined channels have been identified in the rdrp structures that allow access to the active site of the incoming ribonucleoside triphosphate (rntps) and the rna template, as well as the exit path of the newly synthesized daughterstrand rna (dsrna) product (ferrer-orta et al., ) . according to the crystal structure of serotype hrv b solved by love and his coworkers, the finger domain can be divided into an n-terminal segment containing the first residues, an "inner" region surrounding the palm domain, and an "outer" region projecting away from the palm. the n-terminal segment folds back on itself, forming a smaller loop (residues À ), which sits at the top of the polymerase molecule and forms contacts with the tip of the fingers subdomain. this n-terminal segment serves as a bridge reaching across to form interactions with the thumb domain. this bridging of the fingers and thumb subdomains is unique to rdrps and gives them a globular shape rather than the u shape observed in the dna polymerases, ddrps, and the hiv rt. it could be possible that the disruption in the n-terminal region leads to the inactivity of the polymerase. the "inner fingers" region consists primarily of helices α , α , α , α , and α which surround round and pack against the palm domain. the "outer fingers" region includes: ( ) a five stranded β sheet (β , β , β , β , and β ), ( ) an adjacent helix α and β-hairpin β Àβ , and ( ) a loop formed by β and α . strand β containing motif f (residues À ) is unique to the rdrp family (ago et al., ) . this motif f contains residue arg , which is a highly conserved residue of hrv, and interacts with the α-phosphate of the nascent ntp (huang et al., ) . a topological feature of hrv d pol which is unique to picornaviral rdrps is the β-hairpin defined by β Àβ (residues À and À ). differences are observed in the serotype hrv d pol , as compared to hrv b, where a unique small loop (residues À ) is inserted after a short α-helix on the front face of the finger subdomain and motif f comprises residues À . the palm subdomain (residues À and À ) is defined by the central β-sheet β , β , and β surrounded by helices α , α , and α . often referred to as the catalytic subdomain, the palm subdomain is rich in conserved structural motifs, motifs aÀe. motif a (residues À ) is defined by β and α and contains an important conserved . rhinovirus polymerase residue asp , one of the primary magnesium coordination residues required for catalysis. motif a also contains asp , which plays a key role in discriminating between ribonuleotides and -deoxyribonucleotides by hydrogen bonding to the -oh of ntp (hansen et al., ) . motif b (residues À ) includes most of the helix α , along with highly conserved asn , which forms hydrogen bonds to asp and positions the latter for ntp recognition (hansen et al., ) . motif c (residues À ) contains asp , which, along with asp , are absolutely required for the nucleotidyl transfer reaction and are responsible for chelation of two magnesium ions at the active site. motif d provides structural support to motif a. an additional conserved structural element unique to rna-dependent polymerases is motif e (residues À ) that forms a tight loop which lies at the junction between the palm and thumb subdomains. the turn of this loop projects into the active site cavity where it helps to position the end of the primer strand in proper orientation for the attack on the α-phosphate of the ntp during phosphoryl transfer (jacobo-molina et al., ) . similar structural features of the palm domain with motifs aÀe are observed for the other serotypes of hrv. the thumb subdomain of hrv d pol (serotype hrv b) is formed by four long helices α , α , α , α , and the short helix α , all of which superimpose well with pv d pol . the folding topology of the hvr b thumb is similar to the rhdv enzyme, except that the latter lacks the short helix α . structural differences are observed for hrv d pol compared to hrv b, since it consists of three antiparallel α-helices followed by an extended loop segment which connects to an additional c terminal α-helix. the thumb subdomain of hrv d pol differs considerably from that of hcv, bvdv, and ϕ polymerases also. the larger thumb subdomain of hcv ns b contains more than twice the number of residues as compared to hrv dp ol and includes three additional α-helices and a β-loop structure. unlike hcv, where the c-terminal helix fills the active site, the c-terminal helix of hrv d pol packs against the front faces of the molecule in such a way to leave the active site cavity largely exposed (appleby et al., ) . during crystallographic refinement of hrv b d pol , a potential monovalent cation (potassium-binding site) was identified at the interface between the inner and outer fingers domain. the local geometry of this site is found to be preserved in hrv and hrv d pol . hrv b crystallization scheme employs high concentrations of potassium and sodium. it has been observed experimentally that the enzyme's . structural features of hrv polymerase polymerase activity is higher in assay buffer containing potassium ions relative to sodium ions. furthermore, at low concentrations, potassium has a modest stimulatory effect on hrv d pol polymerase activity (hung et al., ) . the proposed potassium-binding site in hrv d pol is about Å from the catalytic residues and does not appear to be directly involved in the polymerase activity or rna binding. potassium ion more likely plays a structural role by stabilizing one end of the finger's domain β-sheet which includes the n terminus). this site is also adjacent to conserved motifs a and b and thus facilitates their positional stability (love et al., ) . the potassium-binding site cannot be predicted properly for pv d pol since this site is present in the disordered region of the enzyme. conserved aspartic acid residues in the polymerase palm domains coordinate the two magnesium ions needed for the catalytic polymerization reaction of the enzyme, with one metal activating the primer oh for the attack of the nucleotide α-phosphate, and the other metal serving to stabilize the triphosphate moiety ( fig. . ). since it is not possible to obtain hrv d pol crystals with the bound divalent cation (mg ) and ntp, hrv d pol is cocrystallized with trivalent sm , where sm is found to occupy a position similar to divalent mg . there occurs coordination by asp and asp from motif c and asp from motif a. an additional electrostatic interaction is found in hrv d pol between the bound sm and asp (distance b . Å ). comparison of hrv d pol structures obtained with and without sm , and comparison of hrv/sm to unbound hrv or hrv b serotypes, shows that the binding of the metal causes a shift of the strand β- (containing asp ) and a parallel strand β- (containing asp ) inward toward the metal (love et al., ) . however, no global conformational change was observed upon binding of a metal at the active site, consistent with the findings from the several hcv ns b structures containing bound metals and ntp (ago et al., ) . mg in the active site plays another role of ensuring substrate specificity apart from its role in the catalytic transfer of the nucleotidyl moiety to the growing rna strand. in the presence of mg , the enzyme displays strong base and sugar specificity and follows the primerdependent activity. however, when mg was replaced by mn , the specificity for ribonucleotides was lost, utilization of deoxnucleotides became possible and primer-independent de novo activity was observed on the poly (c) template (hung et al., ) . the primer-independent synthesis on a heteropolymeric hairpin rna was performed using a copy-back mechanism previously reported for hcv ns b (behrens et al., ) . the optimum mg concentration needed for the activity of hrv d polymerase was . mm which is comparable to the mm required for the activity of pv and emcv d polymerases. it is interesting to note that unlike in hrv serotype, in case of hrv d pol , it is mn that catalyzes both vpg uridylylation and vpgpoly (u) synthesis on a poly (a) template by about -fold over those in the presence of mg . whether or not the stimulatory activity of mn is due to an enhanced binding of the enzyme to the poly (a)-vpg complex is not yet known (gerber et al., ) . the enzyme uses the positive-sense rna template to add the correct ntp molecule to the growing daughter strand. this occurs in the active site where the template and the initial short nucleotide chain known as primer interact to form a duplex. in the hrv d pol duplex mode there are three domains; each of these plays a significant role in supporting this primer-template duplex by interacting with the sugar phosphate backbone. it consists of the palm domain, finger domain, and the thumb domain. hrv residues À and À , which show relatively high mobility in the crystal structure, are located near the expected "entry" of the template strand ( end) and "exit" of the primer strand ( end), respectively. asp is positioned to form a hydrogen bond with the oh group of ntp, which is responsible for the ribonucleotide discrimination within the rdrp family. arg interacts with the triphosphate moiety of ntp. β-hairpin β Àβ interacts with the major groove of the duplex, possibly serving as a "guide" analogous to the role proposed for the β-loop of hcv ns b. in the absence of direct structural evidence relating to how these physiological substrates bind in the exposed active site of hrv d pol , actually the hiv rt ternary complex structure (huang et al., ) , was used as a guide to model this templateÀprimer duplex and an incoming rntp molecule in the active site. oligomerization of protein refers to the interaction of more than one polypeptide chains. this forms the quaternary structure, generally considered to be the highest level of organization within the protein structural hierarchy. oligomeric proteins may be composed either exclusively of several copies of identical polypeptide chains, in which case they are termed homo-oligomers, or alternatively by at least one copy of different polypeptide chains (hetero-oligomers). rdrps can oligomerize through reversible associations mediated by electrostatic and hydrophobic interactions, hydrogen bonds or by covalent stabilization by disulfide bonds. oligomerization is predominantly observed in vitro during crystallization, probably due to the high protein concentration, as well as by other factors like ph or ionic strength of the solution. intracellular accumulation of oligomeric polymerases was observed during viral infection of different rna viruses, including pv, sendai virus, rift valley fever virus, and norovirus, among others. within the crystal lattice of pv d pol , molecules interact through two extensive contact surfaces referred to as interfaces i and interfaces ii (hansen et al., ) , giving rise to higher order structures that have been proposed to explain the oligomerization (beckman and kirkegaard, ) . disruption of contacts forming interfaces i in pv d pol crystals diminishes biochemically detected oligomerization. of the three serotypes of hrvs (hrv , , b) none forms crystal lattice contacts resembling interfaces ii of pv. furthermore, neither hrv nor hrv b d pol show interactions found in interfaces i of pv. however, hrv d pol contains an intermolecular contact similar to the interface i. however, the two salt bridges forming interface i in pv are not present in hrv . it is important to note that the oligomerization phenomenon has not been reported till now for any hrv serotypes. also, there is no current evidence which shows that oligomerization plays any role in hrv functioning (pathak et al., ) , but the study of the oligomerization phenomena in hrv polymerase can help in understanding the protein evolution and higher order complexity associated with it. conformational analysis of protein structure is advantageous as it gives an idea about the flexible and restricted regions of the protein. this flexibility of protein is essential for protein function and the dynamics of these regions can be modulated by allosteric effectors, either activating or inhibiting the biological activity of the protein. an important conformational property of all viral rdrps is their "closed-hand" conformation, as opposed to the "open hand" or u shaped observed in other polynucleotide polymerases. here in case of hrv d pol , this closed conformation is accomplished by interconnecting the fingers and thumb domains through the n-terminal portion of the protein and through several loops protruding from the fingers, named the fingertips, which completely encircle the active site of the enzyme. the closed-hand conformation stabilizes a relatively rigid unit. however, different structural data showed that the thumb domain can rotate a few degrees, relative to the fingers and palm, giving a more open conformation that is found to be the inactive form of the enzyme (ng et al., ) . the recent structure of hcv genotype a rdrp revealed an extreme case of this "open" conformation, in which the contact between the fingertips and thumb is partially disrupted (biswal et al., ) . molecular dynamics simulations were performed by the cameron laboratory on the various picornaviral polymerases including pv, hrv, coxsackievirus b (cvb ), and foot-andmouth disease virus (fmdv) (moustafa et al., ) and the results showed conserved patterns of flexibility. molecular dynamics (md) studies showed that the largest flexibility was located at the fingers domain, whereas the lowest flexibility was concentrated in the palm. the conserved structural motifs involved in primer, template, and/or nucleotide binding (motifs a, dÀf) were all quite flexible, even those located in the palm domain. in contrast, the structural motifs whose primary role is to serve as ligands to the divalent cations required for nucleotidyl transfer (the portion of motif a harboring aspa, the α-helix of motif b containing asnb and motif c) appeared to be much less flexible. all these dynamic elements could be modulated by external effectors, which appear to be effective allosteric inhibitors that could block or disturb the flexibility of these enzymes, ultimately reducing their catalytic function. among all structural movements observed, motif b and the β-loop at its n-terminus in particular, came out to be the new potential druggable targets (garriga et al., ) . the role of rdrp is critical, not only for the virus life cycle, but also for its adaptive potential. the combination of low fidelity of replication and the absence of proofreading and excision activities within the rdrps result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. data obtained revealed that the palm mutations produced the greatest effects on the in vitro nucleotide discrimination. this makes these virus species "quasi-species" where individual members of the virus population have some distribution of point mutations relative to a consensus sequence. limiting the quasi-species variation with a high fidelity polymerase variant could prevent the spread of the virus within a mouse model (pfeiffer and kirkegaard, ) . interestingly, reduction in the fidelity can limit the virus growth, as shown by recent coxsackie virus studies, wherein low fidelity polymerase variants resulted in viruses that replicated well in tissue culture, but then rapidly extinguished and failed to establish persistent infections in animal models (gnadig et al., ) . kinetic studies have suggested that the nucleotidyl transfer chemical step and a conformational change in step ( fig. . ) played an important role in the fidelity checkpoints. motif d plays a unique role in this conformational step and helps in limiting the misincorporation of nucleotide (arnold et al., ) . many polymerases use conformational dynamics of a conserved alpha helix to permit correct nucleotide addition. this alpha helix is missing in structures of rdrps and rts. conserved motif d and specifically the residue lysine in this motif plays the substitute role of the alpha helix in other rdrps. motif d plays an important role in nucleotide addition and step * * step step step step + figure . a diagram showing rna-dependent rna polymerase (rdrp) kinetic mechanism that includes a prechemistry conformational rearrangement (step ) required for catalytic competence of the rdrpÀrnaÀntp complex in the forward (nucleotide incorporation) direction. step is where the conformational changes in motif d occurs. maintaining the fidelity. the lysine residue acts as a general acid, it protonates the leaving pyrophosphate group during the nucleotidyl transfer reaction. this proton transfer reaction, as shown in fig. . , while not essential to catalysis, is found to enhance the rate of nucleotidyl transfer by -to -fold (castro et al., (castro et al., , ). mutation of the motif d lysine leads to a dramatic decrease in the nucleotidyl transfer rate and increases the fidelity of the polymerase reaction, hence controlling the diverse population of the virus (vignuzzi et al., ) . yang and coworkers performed the solution state nmr titration studies for pv to show how motif d acts as a checkpoint by differentiating the binding of correct vs. incorrect nucleotide, thus maintaining the fidelity of the enzyme. motif d is a conserved structural feature in pv and rv rdrps. hence, the result obtained for pv could be extended to rv. in particular, binding of the correct nucleotide produces a large chemical shift change to residue met in accordance with a conformational change in motif d, whereas incorrect nucleotide binding fails to produce such a large chemical shift with respect to residue met , suggesting that closed conformation of the enzyme is not reached when the incorrect nucleotide binds. thus, binding of the incorrect nucleotide appears to perturb the position of motif d and shift the conformational equilibrium away from the closed conformation, which is essential for the active state, i.e., it shifts the equilibrium to the left in step of the . a diagram showing proton transfer reaction. as the transition state of nucleotidyl transfer is approached, the -oh proton (h b ) of primer terminus is abstracted by an unidentified base (b) and the pyrophosphate leaving group of nucleoside triphosphate is protonated (h a ) by the lysine general acid. kinetic mechanism (fig. . ) and hence acts as a parameter in the fidelity checkpoint (yang et al., ) . the reaction conditions for the hrv d- his polymerase were optimized using poly (a) as the template and biotinylated oligo (du) as the primer in nen flash plates (hung et al., ) . the incubation temperature was varied between and c, and the optimum temperature was found to be approximately c. at temperatures between and c, there was a dramatic decrease in activity. using the optimum temperature of c, the ph dependence of rdrp activity displayed a bell-shaped curve with the optimum ph of . . divalent metal ions were absolutely required for activity and the optimum concentration for mg was mm. mn was able to substitute for mg while stimulating the activity by . -fold at the optimum concentration of mm. however, the polymerase activity dropped sharply at mm mn . kcl added at low concentrations (, mm) indicate that the monovalent cation k exhibits a modest stimulatory effect but becomes inhibitory at higher concentrations, with the optimum activity observed at mm. hrv d polymerase shows varying preferences for different homopolymeric rna templates. it efficiently acts on poly (a) and poly (c) templates while exhibiting no activity on poly (g) and poly (u) templates. while the polymerase activity of hrv d pol is primerdependent for all four homopolymeric templates in the presence of mg , primer-independent de novo rna synthesis or terminal transferase activity was observed on poly (c) alone when mg was replaced with mn . this phenomenon was previously observed with pv d polymerase (arnold and cameron, ) , whereas for heteropolymeric hairpin rna, hrv d pol was capable of showing primerindependent synthesis using a copy-back mechanism previously reported for hcv ns b (behrens et al., ) , thus producing a dimer. the michaelisÀmenten constant (k m ) and the catalytic constant (k cat ) values determined for utp (uridine- -triphosphate) as substrate using poly (a) as template were within fourfold of the corresponding parameters previously described for pv d polymerase (arnold and cameron, ) . the higher efficiency of the poly (a) template as compared with poly (c) was mainly shown in the turnover rate of the nucleotide substrate where the k cat for utp was about -fold greater than the k cat for gtp (guanosine- -triphosphate). gtp is a purine nucleoside triphosphate, whereas utp is a pyrimidine nucleoside triphosphate, both acting as substrates during rna synthesis. this further suggests that the polymerase enzyme may be more processive on the poly (a) template compared with the poly (c) template. on the other hand, the k m for gtp is -fold lower than that of utp, suggesting that the number of hydrogen bonds involved in base pairing with the complementary nucleotide on the template may also contribute to efficient binding of the nucleotide substrate to the polymerase complex, where gtp can form three hydrogen bonds whereas utp forms only two hydrogen bonds. we studied the substrate specificity for hrv d polymerase in the presence of either . mm mg or mn by testing the ability of the enzyme to incorporate different nucleotides into nucleic acid products. in the presence of mg , hrv d was accurate in the utilization of only ntps complementary to the template and had no rt activity. in the presence of mg , the enzyme could not polymerize dtmp (deoxythymidine monophosphate), suggesting that the enzyme was devoid of significant rt activity. unlike pv d polymerase, replacement of mg by mn did not affect the base specificity of hrv d, but severely compromised the ability of the enzyme to discriminate the sugar moiety of the nucleotide substrates. when mg was replaced by mn , sugar specificity was compromised, rt activity was stimulated by -fold and misincorporation of dtmp during active rna polymerization was increased by -fold. the hrv genome encodes an rdrp designated as d polymerase ( d pol ) which is required for ( ) replication of the hrv rna genome and also ( ) catalyzes the covalent linkage of ump to a tyrosine residue on a short peptide encoded by b (denoted as vpg) which works as the primer in the replication process. therefore, hrv d polymerase possesses two distinct enzymatic functions. the main function of the polymerase is to copy a template nucleic acid strand to produce a daughter strand. hence, the primary catalytic activity of a polymerase is to transfer a nucleotidyl moiety of an incoming nucleoside triphosphate (ntp) that is complementary to the template strand to the ʹ-oh end of a growing daughter strand of rna or dna. the polymerase active site thus must have binding sites for a template strand, the dna or rna primer terminus (the initiation "i" site), and the incoming ntp (the "i " site). the catalytic reaction consists of the following steps: ( ) binding of templateÀprimer and ntp, ( ) incorporation of nucleoside monophosphate into the growing daughter strand, ( ) release of pyrophosphate, and ( ) translocation along the template. the binding of the templateÀprimerÀntp complex is modeled based on hiv rt ternary complex structure (huang et al., ) , as depicted in the modeling of the duplex in the above section. the second step of nucleotidyl transfer to the growing daughter strand is performed with the help of two divalent metal ions in the active site. both the metal ions would stabilize the charge and geometry of the pentavalent transition state during the nucleotidyl transfer reaction (fig. . ). in the third step, the -oh of the rna or dna primer terminus attacks the α-phosphate of the ntp, and a new phosphodiester bond is formed with a release of pyrophosphate. upon release of a pyrophosphate product, in the fourth step, the newly formed rna or dna primer terminus translocates by one base from the site i to the site i, and this nucleotidyl transfer process is repeated until the whole template strand is copied or a termination signal appears. the reaction steps of the whole catalytic activity can be broadly divided into three steps: initiation, elongation, and termination. unlike cellular dna and rna polymerases, which require oligonucleotides to initiate nucleic acid synthesis, viral polymerases can initiate genome replication using a variety of mechanisms reflecting their adaptation to the host cell. all known polymerases synthesize nucleic acid in the ʹ to ʹ direction and thus initiate the nucleotidyl transfer reaction at the ʹ end of the template strand. polymerases use one of two initiation mechanisms, either a primer-dependent or a de novo (primer-independent) mechanism. primer-dependent initiation mechanism can be performed in four ways (fig. . ). it may require ( ) oligonucleotide priming: a short leader rna (two to five nucleotides) synthesized by viral rdrps during abortive cycling that can act as primer. ( ) cap-snatching: oligonucleotides cleaved from the end of a capped cellular mrna are being used by many segmented negative-strand rna viruses for transcription. ( ) protein priming: with protein-primed initiation, an amino acid provides the hydroxyl group for the formation of a phosphodiester bond with the first nucleotide. this mechanism is used by the family picornaviridae (xiang et al., ) as well as dna viruses such as adenoviruses and bacteriophages w and prd . rv rna polymerase performs its initiation step using this mechanism only. the amino acid used by the enzyme is tyrosine. ( ) copy back: with template primed initiation (also known as loop-back, copy-back, turn-around, or back-priming synthesis), the end of the template rna loops back on itself to serve as a primer. discussing the de novo initiation, also known as primer-independent initiation, requires interactions of at least the following four components: ( ) the rdrp, ( ) the rna template with a virus-specific initiation nucleotide, ( ) the initiation nucleoside triphosphate (d ), and ( ) a second ntp (d ). the first phosphodiester bond is formed between d and d . the initiation nucleotide (essentially a one-nucleotide primer) provides the -oh for the addition of the next nucleotide. de novo rna synthesis is advantageous over the primer dependent one, since no genetic information is lost during replication and no additional enzymes are needed to generate the primer or to cleave the region between the template and the newly synthesized daughter rna. in most cases, the productive de novo initiation event is immediately followed by elongation. however, in some instances, de novo initiation leads to the formation of abortive rna products (abortive initiation) or gives rise to short rna oligonucleotides that are subsequently used as primers (known as prime and realign mechanism). polymerases from picornaviridae that use a primer to initiate nucleotide synthesis have small thumb domains and thus have wider template-binding channels necessary to accommodate both a template and a primer, unlike polymerases that use de novo initiation, which have large thumb domains and thus have narrower template-binding channels that can accommodate only single-stranded rna and ntps. rv rdrp follows a primer-dependent mechanism. under this, it follows a proteinÀprimer pathway. it utilizes the uridylylated form of vpg protein as its primer. the process of uridylyation of vpg is also performed by d pol . de novo initiation of viral rna (vrna) synthesis seems to involve a higher k m for d than for other ntps indicating that initiation is the replication efficiency-limiting step that may be subject to additional regulation. initiation efficiency can be affected by the affinity of the polymerase for the initiation nucleotide of the template (t ) and the initiation ntps (d and d ). for instance, biochemical analysis shows that for bvdv rdrp, the n -and c -amino groups of the initiation template cytidylate are essential for rna synthesis (kim et al., ) . consistently, gtp is the preferred d nucleotide for rdrps of bmv (quadt et al., ) , qb (yoshinari et al., ) , bvdv, hcv, gb virus c (gbv-c), and members of the family cystoviridae. temperature also plays an important role in deciding whether rdrp follows the primer dependent or primer independent mechanism. for example, the recombinant dengue virus rdrp can either initiate rna polymerization using de novo or by extending the template terminus by back priming. at moderate temperatures, the enzyme predominantly uses de novo initiation, whereas back priming dominates at elevated temperatures (ackermann and padmanabhan, ) . based on these results and the observed structural differences between the pv and hcv rdrp, a steric model for dengue rdrp was suggested, where it can exist in either a closed or an open conformation, wherein the open one is favored at higher temperatures. the open conformation binds to a fold-back structure at the terminus of the template with the subsequent elongation producing a dimerized product. conversely, the closed conformation, favored at lower temperatures, recognizes the single-stranded terminus of the template and initiates de novo synthesis. such a temperature dependence has not been studied in case of hrv yet, but possible conformation changes on temperature change can lead to such an observed behavior. in an attempt to gain more insights into rv rna replication, morrow and group studied rdrp from hela cells infected with human rhinovirus type (hrv- ). they found that a partially purified preparation of rv rna polymerase copies vrna without an added primer, whereas a more purified preparation is completely inactive in copying vrna unless it is provided with a hela cell protein (host factor) or a synthetic oligouridylic acid [oligo(u)] primer. this suggests that the host cell protein (or host factor) is essential for the initiation step of rv rna replication (morrow et al., ) . the elongation process of rna replication is divided into three steps: nucleotide selection, phosphodiester bond formation, and translocation to the next nucleotide of the template for the next round of nucleotide addition. the structures of coxsackievirus, rv, and pv polymerases elongation complexes (ecs) are solved by engineering rna-mediated crystal contacts (gong et al., ) . the following points can be inferred regarding ec of rv (hrv ) and the structural similarities that are conserved among all three types. the ec of pv polymerase provided a required view about how the template and the rna strand interact as they thread through the active site and showed that viral rdrps use a unique palm-based structural change to close their active site for catalysis (gong and peersen, ) . a comparison of available rdrp structures further showed that this palm-based mechanism is likely to be used by all the positive-strand rna virus polymerases (including rv) and the authors hypothesized that it evolved because the fingers domains of these polymerases made a key structural contact with the top of the thumb domain that tethered the fingers, precluding swinging motions. within the active site, the templating nucleotide is always found fully stacked on the upstream rna duplex and prepositioned for hydrogen-bonding interactions with the incoming ntp. the next templating nucleotide, i.e., at the position in the downstream direction, is found above the active site where its base moiety is bound in a pocket formed between pro and lys from the index finger. at the product side of the active site strong electron density can be observed for a conserved and noncanonical a-form backbone orientation of the phosphodiester linkage between the À and À nucleotides of the template strand. this conformation is due to buried salt bridges between lys and the À phosphate and between arg and the À phosphate, and together these interactions hold the ribose unit in tight contact with the conserved rdrp motif b located at the junction of the palm and fingers domains. in particular, two residues within motif b of pv, ser, and asn (asn in hrv), and an asp residue (asp in hrv) at the c-terminus of motif a, strictly conserved among picornavirus and caliciviruses, form the ribose-binding pocket. these amino acids interact with the ribose hydroxyl groups of the incoming rntp, thus help in the stabilization of the subtle positioning of the palm domain that results in the conformation favoring the functional active site. an incorrect nucleotide can also bind, but its ribose hydroxyl will not be correctly positioned by these residues for the active site closure; and in consequence of this the incorporation efficiency of incorrect nucleotide will be reduced. proton-inventory experiments performed by castro and group showed that two protons are being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the -hydroxyl nucleophile (h a ) and protonation of the pyrophosphate leaving group (h b ) occur in the transition state for phosphodiester bond formation (fig. . ). these data suggest the existence of a general base for deprotonation of the -oh nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group (which happens to be a lysine residue) in all nucleic acid polymerases (castro et al., ) . it is known that all polymerases synthesize the rna strand in the ʹ to ʹ end. for that, they attack the ʹ oh group of the template. thus, the upstream position is the ʹ end of the rna strand and the ʹ end of template, whereas the downstream position will be the ʹ end of rna and the ʹ end of the template. discussing the downstream and upstream positions in the ec of polymerase enzyme can help in visualizing how the template strand and the complimentary rna strand interact and occupy the space in the enzyme going from ʹ (upstream) to ʹ (downstream) direction of the rna transcription. crystal packing contacts stabilize the orientation of the downstream rna stem-loop in the rv ec. the polymerase index finger and pinky finger regions form the cleft on which the downstream rna duplex rides as thick noodles. the rv ec is the only structure where we observe significant contacts involving the downstream template duplex, due to the fact that the rv helix is held in place via nonspecific interactions with other polymerase in the crystal lattice. in case of pv ec structure, we only observe À base pairs of the downstream helix with sufficient density to allow for model building. the pv structure shows that the downstream helix is rotated by , degree such that the ʹ end of the helix is closer to the thumb domain than the fingers domain, whereas no such rotation is observed in case of rv. on the downstream template side, there is a conserved binding pocket for the nucleotide and this anchoring interaction probably acts in concert with the upstream clamp to establish the high temporal stability of the ecs. the ec features an upstream rna duplex that is pinched between the pinky finger and thumb domains such that the rna exit channel is opened by nearly Å as compared to the polymerase alone structures. due to this tight grip on the exiting duplex between the finger and domain, a high temporal stability is observed and results in high processivity of the picornaviral ecs. after the formation of rdrpÀrnaÀntp ec, the next step is the translocation of the polymerase by base pair to position the next templating nucleotide in the active site for the next round of nucleotide addition. a number of crystal structures have also been solved in a post translocation state for various other viruses (gong et al., ) . however, no structures of translocation intermediates are currently available for any rdrps and the precise mechanism is not yet known. once a stable polymeraseÀnucleic acid complex is formed, the polymerases can add thousands of nucleotides during elongation. high elongation processivity requires a stable association of template and primer in the polymerase, which prevents dissociation, but which should also be nonspecific and weak enough to facilitate the translocation movement upon each nucleotidyl transfer reaction step. for example, pv rdrp can add approximately nucleotides and , nucleotides in the absence and presence of its accessory protein ab, respectively, and can synthesize the entire genome in one single event (rodriguez-wells et al., ) . it is poorly understood how viral polymerases terminate nucleic acid synthesis at the ʹ end of the genome. ending precisely at the ʹ terminus of the genome would be particularly challenging for viruses with linear genomes, such as single-stranded rna or dna viruses, and applied to hrv also since it is a single-stranded rna virus. rna virus uses different mechanisms to perform the critical task of rna replication. in the case of hcv, ns b contains a large thumb subdomain with a β-hairpin extension. evidence suggests that the β-hairpin forms a scaffold upon which a ʹ terminal initiation complex can assemble. a similar de novo initiation mechanism has been proposed for the rdrp from bacteriophage ϕ , but a different mechanism, primer dependent, is employed for pv, rhdv, and hrv in which a small viral peptide, vpg (also known as b), is supposed to play the role of a protein primer in the initiation of rna synthesis. actually, it is the uridylylated form of vpg which acts as a primer, so the process of uridylyation of vpg has to be carried out and it is performed by d polymerase. the covalent attachment of uridine monophosphate (ump) to the hydroxyl group of a tyrosine residue located three residues away from the n-terminus of the small viral protein vpg is called the uridylyation of vpg. vpg, dpol, poly (a), and utp form a complex that allows transfer of ump to the hydroxyl group of residue y of vpg (fig. . ). although the exact molecular mechanism of vpg uridylyation by hrv d polymerase is not known, a few structural aspects are known by correlating with pv polymerase. using the crystal structure of hrv d polymerase, a front loading mechanism has been proposed for the uridylyation (appleby et al., ) . in the complex, the polymerase binds directly to vpg and utilizes a stem-loop cis-acting replication element (cre) as a template to uridylylate the tyrosine residue of vpg protein. this product, vpg-pu, is subsequently extended by at least one more uridine nucleotide to give vpg-pupu. residues: gly , arg , and thr in hrv d pol map to the backside of the polymerase molecule at the junction between the palm and thumb subdomains and are believed to be essential for the efficient binding of ab (precursor of vpg) and uridylylation of vpg. currently, there are no licensed treatments or vaccines for rv infections, although several are in development. pleconaril, a viral capsid inhibitor, has been shown to reduce the rv viral load in respiratory secretions and in shortening of the duration of respiratory symptoms in adults with rv respiratory tract infections (pevear et al., ) . but, it is not licensed for use in the united kingdom and united states. other medications that are under development for the treatment of rv infections include rupintrivir, vapendavir, oc (a crth receptor antagonist), and soluble icam- . due to an incomplete understanding of the immune response to rvs and the large number of genetically distinct rv strains, the search for a suitable rv vaccine has so far proved difficult. thus, studying the life cycle of the virus replication can help us in finding specific enzymes involved that could be targeted for drug designing. the rv life cycle offers many opportunities for intervention. the initial stage of the viral life cycle involves binding to a cell surface receptor, endocytosis, acidification of the endosome, viral uncoating, and release of vrna to the cytoplasm (prchla et al., ) . in the next stage, vrna is translated as a single large polyprotein from an internal ribosomal entry site (ires) which is located in the untranslated region of the rv genome. the polyprotein is then processed through sequential enzymatic cleavages by two virus-encoded proteinases, a and c proteinase (foeger et al., ) . simultaneously, transcription of viral genome that is catalyzed by virus-encoded rna polymerase begins. in the final stage of viral replication, rna and capsid proteins are assembled into mature viral particles, which are then released by cell lysis. during the replication process, rna viruses, including rv, modify the cellular functions to their advantage. for example, rv proteinase a attacks the host translational machinery in susceptible hela cells by cleaving the eukaryotic initiation factor (eif)- gi and eif gii, thereby shutting off cap-dependent host cell protein synthesis (glaser and skern, ; gradi et al., ) . drugs which interfere with any of the above steps would be beneficial in treating the rv infection. here in our study, we focus on the compounds that inhibit the functioning of rna polymerase enzyme. hung and group investigated the effect of zn on the activity of hrv d polymerase. zn was inhibitory at mm when tested in the standard hrv d assay buffer using polya/t as template. the ic values of zn tested against polya/t and sshrna templates were reported. in the absence of dtt, zn has an ic of . À . mm when polya/t was used as template. with sshrna, zn is sevenfold less inhibitory. the inhibition of rv d polymerase by zinc is not likely due to displacement of the catalytically essential mg ion, because the mg optimum concentration is at . mm, while the ic of zn is μm (hung et al., ) . at this point, the mechanism of action by zn at the rna polymerase enzyme is not known. but possible modes of action have been proposed by many authors for the in vitro inhibition of rv replication by zn where it inhibits the capsid polypeptides (korant et al., ) . gliotoxin ( fig. . a), a fungal metabolite, is an important member of the epidithiapiperazinediones class of compounds that inhibit the growth of several animal viruses, including picornaviruses. earlier evidences indicated that it is a selective inhibitor of picornavirus rna synthesis and had less effect on protein synthesis when added later during infection. the concentration of gliotoxin sufficient to block vrna synthesis completely does not affect cellular rna synthesis (trown and bilello, ) . the in vivo inhibitory effect of gliotoxin on hrv d polymerase has an ic value of μm (hung et al., ) . kinetic experiments have also shown that the probable mode of action of this epidithiapiperazinedione is via the inhibition of viral rdrp rather than synthesis of this enzyme (trown and bilello, ) . previously, it has also been reported to inhibit the d polymerase of pv where it potently inhibited the incorporation of [ h] uridine into pv rna soon after its addition to the culture medium (rodriguez and carrasco, ) . in fact, two structural forms of gliotoxin exist, the oxidized (i) and the reduced form (ii), but the inhibitory activity of gilotoxin is attributed to the oxidized form (i) (fig. . ) . this is because an interaction occurs between the inhibitor and protein via formation of a mixed disulfide between the disulfide bridge of the inhibitor and a sulfhydryl group on a protein (targeting the cysteine residue) and such an interaction is unlikely with the dithiol form (reduced form) of the inhibitor and hence the oxidized form is the active form. the prospect of using nucleoside analogs in treating viral infections is a new frontier in designing novel antiviral therapy (deval et al., ) . nucleoside analogs attack the active site of the polymerase and need to be converted by the host cell machinery to the corresponding nucleotides, which can either be incorporated by the viral polymerase into the nascent rna or induce premature termination of rna synthesis (carfi et al., ) . -deaza- ʹ-c-methyl-adenosine, a nucleoside molecule ( fig. . b (olsen et al., ) . in contrast, non-nucleoside inhibitors (nnis) are allosteric inhibitors believed to block the enzyme by preventing a conformational transition needed for the formation of a productive polymeraseÀrna complex. van der linden and group reported an nni, gpc-n ( , -[( -chloro- , -phenylene)bis(oxy)]bis( -nitro-benzonitrile)) ( fig. . c) with broadspectrum activity against enteroviruses and cardioviruses. the genus enterovirus contains four human enterovirus species (hev-a, -b, -c, -d), three hrv species (hrv-a, -b, -c), simian enterovirus, bovine enterovirus, and porcine enterovirus. all rvs included in the study (hrv , hrv ) were found to be sensitive to the inhibitory effect of gpc-n , with the ec values ranging from . μm to . μm. in vivo analysis showed that the inhibitor targeted the activity of rna polymerase ( d pol ). the analysis of the difference in the electron density maps revealed the presence of extra densities to position the inhibitor within a pocket located at the bottom of the template channel, mimicking the position of the template acceptor nucleotide, the nucleotide which will base pair with the incoming nucleotide. hence, both the crystallographic studies as well as the order-of-addition experiment strongly suggest that the gpc-n affected the binding of the templateÀprimer in the active site of d pol (van der linden et al., ) . quercetin (fig. . d ), and its derivative -methylquercetin (castrillo and carrasco, ) were shown to inhibit the synthesis of pv rna and this was attributed to the inhibition of vrna polymerase d pol (neznanov et al., ) . in the case of rv, quercetin interferes with the processing of rv polyprotein by rv proteases and this process is required for activation of rna polymerase ( d pol ). results demonstrated that quercetin significantly reduced both negative-and positive-strand vrna and it is due to reduced cleavage of eifg ii and reduction in viral capsid protein, vp , suggesting that quercetin actually targets the initial polypeptide processing that in turn is required for both vrna polymerase processing and for cleavage of eifg ii. another possibility could be that it may directly inhibit vrna polymerase, thereby blocking genome translation and synthesis of new progeny virus (ganesan et al., ) . the rdrp first copies input positive-strand rna (viral genome) into negative-strand rna, which, in turn, serves as a template for the synthesis of viral progeny positive strands from a multistranded replicative intermediate (ri) . the possible inhibitory effect of -fmc ( fig. . e) in case of hrv serotype was examined by an in-house developed quantitative rv-specific rt-pcr elisa method (lauwers et al., ) . the results showed that -fmc exerts its antiviral action by inhibiting the vrna synthesis step during hrv replication in cells. although the exact viral target protein is not known in this case, but it provides an opportunity to find the exact antiviral target of -fmc in the vrna synthesis steps using escape mutants selected against -fmc (verheyden et al., ) . recently, pyrrolidine dithiocarbamate (pdtc) (fig. . f), has been described as a potent inhibitor of hrv multiplication. pdtc is effective against all tested hrv serotypes in several cell lines analyzed, without being toxic (gaudernak et al., ) . in a study performed by krenn and coworkers, the data indicated that pdtc hampers the processing of the hrv polyprotein, thus targeting the enzyme viral proteases a pro and/ or c pro (krenn et al., ) . pdtc also chelates various metal ions, leading to the formation of lipophilic dithiocarbamateÀmetal complexes (thorn and ludwig, ) and can thus promote cell entry of metals, e.g., diethyldithiocarbamate in the form of these lipophilic metal complexes. pdtc is found to increase the intracellular copper levels in thymocytes (erl et al., ) . kim et al. ( ) demonstrated that pdtc can also increase the uptake of zinc ions, and it is known that zinc ion inhibits the activity of the d pol of hrv in vitro (hung et al., ) . thus, it cannot be ruled out that apart from targeting protease enzyme, pdtc could also target the rna polymerase enzyme ( d pol ) function by increasing the intracellular zinc ion concentration (krenn et al., ) . (t- ; -fluoro- -hydroxy- -pyrazinecarboxamid, fig. . g) is an antiviral drug that selectively inhibited the rdrp of influenza virus. it showed specific activity against all three influenza a, b, and c (furuta et al., ) . it also inhibited the rv replication in hela cells, with an ec of μg/ml (furuta et al., ) . analysis showed that the primary mechanism of action of favipiravir against the influenza virus was specific inhibition of vrna polymerase (furuta et al., ) . it is predicted that a similar mechanism might occur with other viruses, such as pv and rv, inhibited by favipiravir, which may account for its broad-spectrum inhibition. mechanistic studies show that the favipiravir and its form favipiravir-rmp (favipiravir-ribofuranosyl- -monophosphate) do not inhibit influenza rna polymerase activity, but it is the phosphoribosylated form, favipiravir-ribofuranosyl- triphosphate (rtp) that inhibits the enzyme. metabolism of favipiravir to its triphosphate form occurs in an extracellular environment in a concentration-dependent manner. the vrna polymerase mistakenly recognizes favipiravir-rtp as a purine nucleotide. this favipiravir-rtp is misincorporated in nascent vrna, or it may act by binding to conserved polymerase domains, preventing incorporation of nucleotides for vrna replication and transcription (jin et al., ) . in this work we have given detailed recent studies on the structure, function, and inhibitors of the rv rna polymerase. crystal structures of the serotypes hrv b, , and have been recently determined. not all serotypes have been studied extensively and an immense scope is still left for such crystallographic studies. rna polymerase performs the dual function of rna replication and uridylation of vpg. the mechanism of this uridylation process is still not fully understood. also, various steps of replication processes are only modeled based on the pv and hiv-rt d pol . very little information is known about the termination step. these gaps need to be filled with further advancements using the techniques previously reported for other viruses of the picornaviridae family. the more advance picture of the enzyme can help in bringing more specific antirhinoviral drugs. currently no drug targeting the rv polymerase is approved to be clinically used to treat rv infection. hence, drug design targeting this enzyme needs more attention of both experimental and theoretical chemists. hopefully, this chapter may provide some guidelines in this direction. de novo synthesis of rna by the dengue virus rna-dependent rna polymerase exhibits temperature dependence at the initiation but not elongation phase crystal structure of the rna-dependent rna polymerase of hepatitis c virus two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity crystal structure of complete rhinovirus rna polymerase suggests front loading of protein primer poliovirus rna-dependent rna polymerase ( dpol) is sufficient for template switching in vivo remote site control of an active site fidelity checkpoint in a viral rnadependent rna polymerase site size of cooperative single-stranded rna binding by poliovirus rna-dependent rna polymerase identification and properties of the rnadependent rna polymerase of hepatitis c virus crystal structures of the rna-dependent rna polymerase genotype a of hepatitis c virus reveal two conformations and suggest mechanisms of inhibition by nonnucleoside inhibitors in vitro tests to evaluate immunotoxicity: a preliminary study action of -methylquercetin on poliovirus rna replication two proton transfers in the transition state for nucleotidyl transfer catalyzed by rna-and dnadependent rna and dna polymerases nucleic acid polymerases use a general acid for nucleotidyl transfer viral polymerases. viral molecular machines inhibition of viral rna polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand rna viruses beyond hepatitis c virus pyrrolidine dithiocarbamate induced apoptosis depends on cell type, density, and the presence of cu and zn a comparison of viral rnadependent rna polymerases human rhinovirus apro recognition of eukaryotic initiation factor gi involvement of an exosite in vitro and in vivo activities of anti-influenza virus compound t- mechanism of action of t- against influenza virus favipiravir (t- ), a novel viral rna polymerase inhibitor quercetin inhibits rhinovirus replication in vitro and in vivo role of motif b loop in allosteric regulation of rna-dependent rna polymerization activity antiviral effects of pyrrolidine dithiocarbamate on human rhinoviruses biochemical and genetic studies of the initiation of human rhinovirus rna replication: purification and enzymatic analysis of the rna-dependent rna polymerase dpol extremely efficient cleavage of eif g by picornaviral proteinases l and a in vitro structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering rna mediated crystal contacts human rhinovirus a proteinase cleavage sites in eukaryotic initiation factors (eif) gi and eif gii are different the major human rhinovirus receptor is icam- structure of the rna-dependent rna polymerase of poliovirus members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus structure of a covalently trapped catalytic complex of hiv- reverse transcriptase: implications for drug resistance biochemical characterization of rhinovirus rnadependent rna polymerase crystal structure of human immunodeficiency virus type reverse transcriptase complexed with double-stranded dna at . Å resolution shows bent dna the ambiguous basepairing and high substrate efficiency of t- (favipiravir) ribofuranosyl -triphosphate towards influenza a virus polymerase pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level template nucleotide moieties required for de novo initiation of rna synthesis by a recombinant viral rna-dependent rna polymerase updates in the relationship between human rhinovirus and asthma zinc ions inhibit replication of rhinoviruses inhibition of polyprotein processing and rna replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype that caused influenza-like illness in new york state during screening of an enterovirus specific rt-pcr elisa method for the quantification of enterovirus genomes in human body fluids by means of a three-level experimental design crystal structure of the rna-dependent rna polymerase from hepatitis c virus reveals a fully encircled active site the crystal structure of the rna-dependent rna polymerase from human rhinovirus: a dual function target for common cold antiviral therapy the tecumseh study of respiratory illness. ii. patterns of occurrence of infection with respiratory pathogensm À purification of a soluble template-dependent rhinovirus rna polymerase and its dependence on a host cell protein for viral rna synthesis molecular dynamics simulations of viral rna polymerases link conserved and correlated motions of functional elements to fidelity quercetinase pirin makes poliovirus replication resistant to flavonoid quercetin crystal structures of active and inactive conformations of a caliciviral rnadependent rna polymerase a -deaza-adenosine analog is a potent and selective inhibitor of hepatitis c virus replication with excellent pharmacokinetic properties structure-function relationships of the rna-dependent rna polymerase from poliovirus ( d pol ). a surface of the primary oligomerization domain functions in capsid precursor processing and vpg uridylylation possible unifying mechanism of picornavirus genome replication relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice rhinoviruses: important respiratory pathogens uncoating of human rhinovirus serotype from late endosomes characterization of a host protein associated with brome mosaic virus rna-dependent rna polymerase gliotoxin: inhibitor of poliovirus rna synthesis that blocks the viral rna polymerase dpol primer-dependent synthesis by poliovirus rna-dependent rna polymerase ( dpol) the dithiocarbamates and related compounds isolation of a receptor protein involved in attachment of human rhinoviruses mechanism of action of gliotoxin: elimination of activity by sulphydryl compounds the rna template channel of the rna-dependent rna polymerase as a target for development of antiviral therapy of multiple genera within a virus family mode of action of -furylmercury chloride, an anti-rhinovirus compound engineering attenuated virus vaccines by controlling replication fidelity rna signals in entero-and rhinovirus genome replication motif d of viral rna-dependent rna polymerases determines efficiency and fidelity of nucleotide addition cca initiation boxes without unique promoter elements support in vitro transcription by three viral rnadependent key: cord- -jy j sh authors: huang, ting; wang, wei; bessaud, mael; ren, peijun; sheng, jun; yan, huajie; zhang, jing; lin, xin; wang, yongjin; delpeyroux, francis; deubel, vincent title: evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jy j sh background: human rhinoviruses (hrvs) are a highly prevalent cause of acute respiratory infection in children. they are classified into at least three species, hrv-a, hrv-b and hrv-c, which are characterized by sequencing the ′ untranslated region (utr) or the vp /vp region of the genome. given the increased interest for novel hrv strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of hrv strains in a -year study of children with acute respiratory infection visiting one district hospital in shanghai. methodology/findings: we cloned and sequenced a -nt fragment that covered part of the ′utr and the vp /vp capsid genes. sixty-four hrv-infected outpatients were diagnosed amongst children with acute low respiratory tract infection. two samples were co-infected with hrv-a and hrv-b or hrv-c. by comparative analysis of the vp /vp sequences of the hrvs, we showed a high diversity of strains in hrv-a and hrv-b species, and a prevalence of . % of strains that belonged to the recently identified hrv-c species. when analyzing a fragment of the ′ utr, we characterized at least two subspecies of hrv-c: hrv-cc, which clustered differently from hrv-a and hrv-b, and hrv-ca, which resulted from previous recombination in this region with sequences related to hrv-a. the full-length sequence of one strain of each hrv-ca and hrv-cc subspecies was obtained for comparative analysis. we confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the a gene and ′utr of the hrv-ca strain. double or triple infections with hrv-c and respiratory syncytial virus and/or bocavirus were diagnosed in . % of the hrv-infected patients, but no correlation with severity of clinical outcome was observed. conclusion: our study showed a high diversity of hrv strains that cause bronchitis and pneumonia in children. a predominance of hrv-c over hrv-a and hrv-b was observed, and two subspecies of hrv-c were identified, the diversity of which seemed to be related to recombination with former hrv-a strains. none of the hrv-c strains appeared to have a higher clinical impact than hrv-a or hrv-b on respiratory compromise. human rhinoviruses (hrvs) are a highly p revalent cause of the acute respiratory infection (ari) defined as the common cold [ , , ] , which is frequently associated in children with bronchitis, bronchiolitis, wheezing, pneumonia, asthma and otitis [ , , , , , ] . hrvs are classified in genus enterovirus (hevs) in family picornaviridae [ ] . hrvs are non-enveloped, single-stranded, positive-sense rna viruses of approximately nt, composed of a untranslated region (utr), followed by a long open reading frame coding for capsid proteins vp , vp , vp and vp , and seven non-structural proteins a, b, c, a, b, c and d, and terminated by a short utr and poly a tract. more than serotypes of hrv are known, which have been classified into two species, hrv-a and hrv-b, according to comparative alignment of nucleotide fragments of vp [ , ] , vp /vp [ ] and utr [ , ] , and more recently, on complete genome nucleotide sequences [ ] . moreover, some genomic sequences have been found not to cluster with hrv-a and hrv-b species, which suggests the existence of other species (hrv-c and hrv-d) [ ] . a new species of hrv-c was recently identified worldwide by comparative analysis of vp or vp /vp genes [ , , , , , ] and utr [ , ] . however, discrepancies have appeared in the classification of some of the new hrv-a or hrv-c strains, depending on the size and location of the nucleotide sequence in the viral genome and on the phylogenetic methods used for direct analysis of hrv sequences [ , , , , , , , , , ] . these recent data underline the lack of knowledge about the biodiversity of hrv strains and their worldwide distribution [ , ] . moreover, little is known about the characteristics and diversity of hrvs circulating in a given area in a short period of time. in the present study, we looked for hrvs in a -year collection of nasopharyngeal swabs (npss) of children with ari visiting a district hospital in shanghai, and compared sequences in two regions previously defined for genetic classification of hrv serotypes [ , ] . our study showed a high diversity of hrv species and genotypes, and a prevalence of the novel hrv-c species in npss of children with bronchitis and pneumonia. this biodiversity appeared to result partly from recombination events in the utr, between hrv-c strains and those close or similar to hrv-a species, which led to the suggested classification of hrv-c into at least two subspecies. eight hundred and twenty-seven samples were collected from a group of children consulting the shanghai nanxiang hospital during a -year period, and tested for respiratory viruses using a multiplex rt-pcr (mrt-pcr). sixty-four samples ( . %) were positive for hrv, according to the length of the amplified fragment in the vp /vp region visualized on agarose gel (data not shown). a larger fragment of nt, including part of the utr (starting at nt ) and the vp /vp genes (ending at nt ), was amplified and cloned into plasmid vectors for genetic analysis (table ) . only one sample, n , could not be amplified and was amplified in two steps in the utr (nt - ) and in the vp /vp region (nt - ), respectively. analyses of different clones for each sample allowed the identification of multiple infections: sample n was shown to contain one hrv-a and one hrv-b (n a and n b, respectively), while n contained one hrv-a and one hrv-c (n a and n c, respectively) (see below). in order to further characterize the hrvs identified in the samples, the nucleotide sequences located in the utr ( nt) and the vp /vp genes ( nt) were chosen to allow comparative alignment with sequences of reference serotypes and field strains available in genbank. we first compared and classified shanghai strains according to their vp /vp sequences. the pairwise nucleotide divergence in the vp /vp region ranged from to . %. twenty-seven hrvs ( . %) showed . % nucleotide identity with the closest hrv-a clusters, and five hrvs ( . %) showed . . % nucleotide identity with hrv-b clusters ( table ). the remaining strains ( . %) diverged from hrv-a and hrv-b species by . . % in their vp /vp nucleotide sequence ( table ) . these strains showed from . to % nucleotide identity with each other and were related to the recently described hrv-c strains, nat and nat , isolated in california, usa [ ] , c , c and c in hong kong [ ] , and qpm in australia [ , ] ( fig. ). strains n , n and n were closely related to the recently identified strains c and nta with . . % nucleotide identity, whereas the remaining hrv-c strains showed only . - . % nucleotide identity with six other recent strains (qpm, nat , nat , c , c and c ; table ), which were classified tentatively as hrv-c species [ , , , ] . classification of the strains into three different species was also demonstrated by construction of a phylogenetic tree using aligned vp /vp sequences (fig. ) . to characterize and classify further the chinese hrv strains, utr sequences were considered (table , fig. ). they were compared to the utr of all reference hrvs, to those of new strains identified in children with respiratory illness in wisconsin (indicated as w) [ ] , and to those of other recently identified hrv-c strains [ ] . pairwise nucleotide divergence between the three hrv species was . - . %, and a limit of , % divergence between genotype pairs was chosen for similar genotype assignment in one species [ ] . new genotypes were identified when they had - % pairwise nucleotide divergence from the nearest serotype in the same species (table ) . fifty-five hrvs shared . . % nucleotide identity with strains already identified, and hrvs showed . - . % nucleotide divergence with the nearest known hrvs. they may represent newly discovered genotypes. these strains clustered with hrv-a (n ) or hrv-c species (n , n , n , n , n , n , n , n , n and n ) ( table ) . most surprisingly, of the strains classified as hrv-c by comparative analysis of vp /p sequences ( table ) were related more closely to hrv-a strains when their utrs were analyzed, and showed incongruent clustering in phylogenetic trees (figs. and ). the nucleotide sequences in the utr of these strains were related closely to those of formerly identified qpm, nat , nat , c , c and c hrv-c strains, and to some of the w strains recently identified as hrv-a [ ] (fig. ) . however, our strains clustered together with these six strains in two major branches in the phylogenetic tree constituted a subspecies of hrv-c called hrv-ca (table , figs. and ) . the fourteen other hrv-c strains formed another unique branch of hrv-c subspecies, called hrv-cc, which clustered differently from other species of hrv-a and hrv-b, and from subspecies hrv-ca in the phylogenetic tree based on utr sequences (table ; figs. and ). these strains were related closely to some w strains of hrv that were classified as hrv-c [ ] (fig. , table ) in order to characterize more precisely the differences observed in the utr between hrv-ca and hrv-cc subspecies, and to localize possible recombination sites in the utr of the genome of hrv-ca subspecies, bootscanning and similarity plot analyses were conducted in the gene fragment of nt that included the utr and adjacent capsid genes. hrv-ca nucleotide sequences were scanned against sequences of n , r and r , which are considered as representative strains of hrv cc subspecies and hrv-a and hrv-b species, respectively. stretches of nucleotide sequences that were closer to hrv-a (r ) than to hrv-cc (n ), flanked by sequences related to hrv-c could be detected in the utr of hrv-ca strains, as exemplified with hrv-ca n strain ( fig. a and b) . these hrv-a-related nucleotide stretches were thus flanked by putative recombination sites. these sites were located differently among the hrv-ca strains, delimiting hrv-a-related stretches that ranged from to nt in length (fig. c ). while variable among the strains, the identified recombination sites were all located inside the utr and none of them was identified in the downstream vp /vp coding sequence. hrv-a-related nucleotide sequences and putative recombination sites were also found in the utr of the previously described hrv-c strains c , c , c , nat , nat and qpm (fig. ) . the results corroborated the clustering observed in the phylogenetic tree based on utr sequences (fig. ) , since strains gathered in the same hrv-ca subcluster (for example n , n , n and n , or n , n , n , n , n and n ) displayed the same recombination pattern. these subclusters revealed different recombinant lineages, each of which originated from independent recombination events. in order to further characterize the genome of the hrv-cc subspecies, for which no full-length sequence was yet available, we sequenced the remaining genes that covered the whole coding sequence and utr of n strain, which was chosen as the representative of this subspecies (tables and ). the full-length n genome sequence was compared to those of the hrv-ca subspecies strains c , c and c , and to that of n strain, which was sequenced as the representative of the hrv-ca subspecies. the genome sequences were also compared to those of the hrv-a strains n and r , and to the hrv-b strains r and r (table ). the full-length nucleotide sequence of n strain contained nt, excluding the poly(a) tract, which was shorter than sequences from hrv-a and hrv-b strains, but similar to those of hrv-ca strain n and other related strains (c , c and c ). the aa lengths of the polyprotein and of each of the individual proteins of n were slightly different from those of hrv-a and hrv-b species, but similar to those of other hrv-c strains. the most divergent amino acid length between hrv was observed for the vp protein that was shorter in hrv-c species ( table ). the unique putative cleavage (m/s) site between vp and vp protein identified previously for qpm, c , c and c strains [ ] was also observed for n and n strains. it was different from those of the hrv-a strains n and r (q/s), and from those of the hrv-b strains r and r (n/s) (data not shown). alignment of the vp amino acid sequence of hrv-cc strain n with those of other hrv-a and hrv-b species and hrv-ca subspecies, designated in table , showed structural features typical of hrv-c species [ , , ] (data not shown). in particular, footprints including deletions in the bc, de and hi loops and conserved amino acids potentially involved in inter-cellular adhesion molecule (icam- ) receptor binding [ , , , ] were conserved within the hrv-c species (data not shown). bootscanning and similarity plot analysis conducted on the genomic sequences of n (hrv-ca), n (hrv-cc), r (hrv-a) and r (hrv-b) confirmed that n featured a utr sequence that was related to the r sequence (stretch i), followed by a capsidic sequence related to the n sequence. n nonstructural sequence ( a to utr) was related more closely to n than to r and r sequences. however, in stretch ii (nt - according to n numbering), n strain (hrv-ca) was closer to r (hrv-a) than to r (hrv-b) or n (hrv-cc), which resulted in high bootstrap values between n and r a sequences (fig. ) . this may have been the result of a recombination event that occurred in the part of the a-encoding sequence of the parental strain n , and which involved an hrv-a strain. nevertheless, the hrv-a parental strain or ancestral strain could not be identified since the closest hrv-a a nucleotide sequence available was , % identical to that of n in this region. in contrast from nt , to the end (stretch iii in fig. ) , the n strain genome was found to be closely related to that of n , with nucleotide identity . %. this result is corroborated in figure , which shows a phylogenetic analysis of the utr sequences of n and n compared to those of hrv-ca subspecies and hrv-a and hrv-b species. this suggests that n and n strains share a common recent ancestor through recombination. and ) and on local recombination in utr (see fig. ). b strains closely related with more than % identity. c these strains clustered differently when based on vp /vp sequences (see table and figs. and ) . doi: . /journal.pone. .t clinical outcome from hrv strains isolated from pediatric outpatients among the pediatric patients, were males and females, and their age ranged from months to years. the majority of hrv infections were diagnosed between and years of age ( . %). bronchitis ( . %) and pneumonia ( . %) were highly prevalent in children with comparable incidence in hrv-a and hrv-c infections (table ) . moreover, the ratio of pneumonia over bronchitis ( . %) was comparable to that in the whole cohort of children ( . %). only one child among the hrv-positive patients had asthma and was co-infected with hrv-c, influenza a virus (iav) and respiratory syncytial virus (rsv) ( table ) , whereas of the patient were diagnosed with asthma. hrvs were isolated throughout the years, with a predominance of hrv-c viruses in the cold season (table ) . interestingly, different hrv genotypes were detected within the same period (for example, n and n , n and n /n , n /n and n , and n /n and n /n ), with a larger diversity and distribution of individual or paired hrv-a genotypes compared to hrv-c strains, which clustered in closely-related genotypes (fig. , tables and ) . conversely, n and n strains of samples collected at months interval showed . % identity (fig. ) . single hrv infection was diagnosed in children and coinfections were identified in patients (table ) , with double and five triple infections. the viruses most often identified in hrv co-infection were rsv (six cases) and human bocavirus (hbov; four cases), and two patients were co-infected with hrv, hbov and rsv (table ). there was no difference between hrv-ca or hrv-cc subspecies and any of the clinical or epidemiological data (data not shown). in this report, we looked for hrvs in a -year collection of npss from children with ari visiting a district hospital in shanghai, and found a high diversity of hrv strains that belonged to different species and genotypes. we characterized by rt-pcr and sequenced hrvs, among them hrv-a, five hrv-b, and hrv-c strains. when sequencing the vp /vp region of the hrv genome, several recent studies have identified new strains of viruses from children and adults with ari, asthma, or otitis, which are clustered differently from hrv-a and hrv-b, and have been classified into a novel hrv-c species [ , , , , , , , , , , ] . other groups have also identified novel hrv-c strains by sequencing the vp gene [ ] or the utr [ , ] . the different sizes and locations of the regions amplified in the hrv genomes renders difficult comparative genetic analysis. recently, palmenberg et al. ( ) have finalized the full-length genome sequences of all hrv-a and hrv-b reference strains, and identified structural features of these two species and the novel hrv-c species [ ] . in our study, we identified hrvs ( . %) that clustered differently from hrv-a and hrv-b in a phylogenetic tree that was established on the basis of vp /vp sequences, which were related to recent strains classified in the novel hrv-c species (fig. , table ). fourteen hrv-c strains ( . %) segregated from the other strains ( . %) that were closely related to hrv-a in their utr sequence (fig. ) . this led us to propose a classification of two hrv-c subspecies, hrv-cc and hrv-ca. in previous studies [ ] . these strains clustered with our field strains within the hrv-cc subspecies. moreover, strains that clustered with hrv-a, and had - % pairwise nucleotide divergence from the nearest reference serotype [ ] , clustered within the two major branches of hrv-a and hrv-ca strains (fig. ) . therefore, we cannot ensure that some of the strains were hrv-a or hrv-ca strains. kiang et al. ( ) have identified five novel hrvs out of clinical samples ( . %), which segregated from hrv-a and hrv-b, and were classified as hrv-c, and three additional strains ( . %) that also clustered with qpm, c , c , c , nat and nat [ ] (fig. ) . however, the field hrv strains of these previous studies were sequenced using a utr that did not match fully our sequence and that of lee et al. ( ) [ ] , and could not be included in the present study for comparative analysis. interestingly, the five strains identified in california in [ ] and n and n from our study were closely related to strain w isolated in wisconsin in the late s [ ] , and to nat isolated in the winter of in california [ ] , which confirms that similar genotypes of hrv-ca are widespread [ ] . the strains of hrv-c species identified in the present study were characterized by analyzing the utr, vp , and part of vp (fig. ) . this approach showed the advantages of covering only ncr, vp /vp , vp or d genome fragments. analyzing sequences that covered the utr and the downstream vp / vp capsid region allowed identification of co-infections when several clones were sequenced, and helped to locate the recombination sites in strains of the hrv-ca subspecies. thus, this region of the genome may be useful for building a database of the novel strains that are circulating worldwide. the genome of hevs is subject to frequent recombination [ , , , , , , ] , with interspecies exchanges observed in the utr [ ] . palmenberg et al. ( ) have observed intraspecies recombination in three hrv-a, with structural characteristics and phylogenetic evidence that suggests a novel clade d classification [ ] . tapparel et al. ( ) observed phylogenetic incongruities in utr, vp and cd sequences of two clinical isolates of hrv-a related to recombination [ ] . we observed incongruent clustering of n , n and n strains of hrv-a species in the phylogenetic trees based on the utr or the vp /vp regions of their genomes (figs. and , table ), which suggests intraspecies recombination in the utr. we observed one co-infection with hrv-a and hrv-b (n a and n b), one with hrv-a and hrv-c (n a and n c), and three co-infections of hrv-a and hrv-b with hevs that may favor recombination events. previous comparison of genome sequences between hrvs showed only limited recombination events and a pattern of genetic diversity lower than that observed with other picornaviruses [ ] . the presence in hrv-c subspecies of sequences that share . - . % identity with hrv-a strains (table ) suggests that recombination events occurred between hrv-c and hrv-a. bootscanning of the utr of hrv-c strains also showed different sites and lengths of recombination (fig. c) , which suggested that there were several independent events that led to several groups of hrv-ca genotypes, which formed clusters in the phylogenetic tree (fig. ) . comparative analysis of the full-length nucleotide sequences of two field strains of different hrv-c subspecies (n and n ) with those of other hrv species suggested that multiple interspecies recombination events occurred in the utr and in the ns a protein gene, and that recombination also occurred in the utr between n and a strain close to n . these findings are in agreement with those observed for other hevs, for which recombination events in the capsid-encoding sequence are very rare, probably because of structural constraints that restrict the functioning of chimeric capsids [ ] . this result appeals for the full-length genome sequencing of the major representatives of the hrv-c species, in order to establish a clear understanding of the evolution and classification of the novel virus into subspecies. comparison of the coding sequences of n hrv-cc with other strains of hrv-ca subspecies [ ] , including our field strain n , showed high similarities in the lengths of the proteins, their cleavage sites, and the structural features of vp . these characteristics and the absence of growth in cell culture, noted in our laboratory and by others (data not shown), support the classification of the novel strains into a unique hrv-c subspecies. our clinical specimens all originated from npss from pediatric outpatients. the remarkable outcome of the study is the large diversity of genotypes that has circulated in a relatively small group of people in a district of shanghai during a -year observation. although some clusters of similar genotypes in a limited period of time were observed, co-circulation of different genotypes and hrv species and subspecies, and co-infections with two hrv species were observed. the prevalence of the novel hrv-c in our specimens ( . %) differed from previous studies that associated the prevalence of the novel variant with severe disease outcomes, which ranged from influenza-like illness or infection of the low respiratory tract [ , ] to asthma exacerbation, bronchiolitis, and febrile wheeze [ , , , , , , , ] . all our patients showed bronchitis or pneumonia, with no etiological correlation with any of the species or subspecies of hrv. only one patient co-infected with hrv-c, iav and rsv was diagnosed with asthma among the hrv-positive patients ( . %), whereas of the children had asthma ( %). the difference observed with previous studies, . % [ ] and % [ ] asthma in hrv-positive patients, may be related to the criteria for enrolment. moreover, none of the patients in our study were hospitalized, which makes comparison with hospitalized children difficult [ , ] . another criterion to consider in the trend to correlate clinical symptoms with hrv infection is the presence of co-infecting pathogens. in our study, four strains of hbov and six strains of rsv ( . %) were identified in association with hrv-c ( . %). hbov and rsv are common viruses diagnosed in ari, which are often associated with hrv [ , ] , and hbov was identified in . % of children co-infected with hrv [ ] . nevertheless, the incidence of hbov in ari and in severe outcomes remains elusive [ ] . more studies need to be carried out on large numbers of samples from severe and mild diseases, to identify any obvious role of hrv sequence diversity and association with other pathogens in disease severity. since a large diversity of recombination in hrvs has become obvious, we must be aware of the occurrence of novel hrvs that may become highly virulent. this study was approved by the ethical committee of shanghai nanxiang hospital and written informed consent was obtained from the parents of the children. clinical specimens (n = ) from npss were collected from children under years old, who experienced a lower respiratory tract infection, and who were consulting the pediatric department of shanghai nanxiang hospital during the period october to october . total rna was extracted from nps specimens using qiaamp viral rna mini kit (qiagen, hilden, germany), and stored at uc. rna was amplified using the qiagen one step rt-pcr kit. a five-tube mrt-pcr was used for virus identification as previously described [ , ] . tube targeted iav, influenza b virus, rsv, and human metapneumovirus; tube , parainfluenza viruses to ; tube , hrv and influenza c virus; tube , human coronaviruses (hcovs) e-hcov, oc -hcov, nl -hcov and hku -hcov; and tube , adenovirus and hbov. amplified products were analyzed in . mg/ml ethidium bromide/ % agarose gel. samples that showed positive results for hrv were amplified again using specific primers p - f and vp / r, located in the utr and vp gene, respectively (table ) . one strain of hrv-c (n ) could not be amplified using the p and vp extreme primers and was amplified using primers in utr and vp / vp , respectively (table ). in brief, . ml of extracted rna was mixed with buffer and . mm dntps, . mm of each of the primers, and ml of enzyme mix, and diethylpyrocarbonatetreated ultrapure water was added to a final volume of ml. amplification programs included reverse transcription at uc for min, inactivation at uc for min, followed by cycles at uc for s, uc for s, uc for s, and final extension at uc for min. the amplified dna products were detected by ethidium bromide-agarose gel electrophoresis. the lengths of p -vp , vp -vp and p -p amplicons were , and nt, respectively. dna products were extracted from agarose gels by using qiaquick gel extraction kit (qiagen), and were ligated into pmd -t vector (takara biotechnology, dalian, china), and at least two recombinant plasmids were sequenced in biosune sequence company and life biotechnology in shanghai, china. sequences of different clones of n and n showed identities for either hrv-a or hrv-b strains. more plasmids were sequenced for these strains to confirm that the two patients were originally coinfected with two different hrv species. sequences of three complete genomes of hrv were obtained for strains n (reference r collected on december , ) , n (r collected on june , ) and n (r collected on july ). primers used for the amplification of viral genomes were designed after multiple alignments of sequences from the genomes of different hrvs available in genbank (table ) . overlapping amplified dna products were obtained after pcr of cdna that was obtained using oligodt and a transcriptor high fidelity cdna synthesis kit (roche, mannheim, germany), following the manufacturer's protocols. briefly, . ml viral rna was mixed with ml oligodt and heated at uc for min, and then kept on ice for min. after addition of ml buffer, . ml protector rnase inhibitor, ml dntps, ml dtt, and ml rt enzyme, the reaction was incubated at uc for h, inactivated at uc for min, and stored at uc. amplification of a d region of n , n and n hrv strains was carried out by nested-pcr using takara extaq (takara biotechnology) and specific primers (table ) , for cycles of s at uc, s at uc, and s at uc. to amplify vp (upstream of a) sequences of n and n strains, nested pcr was carried out using takara lataq with gc buffer i(takara biotechnology), specific primers (table ) , and incubation for cycles of s at uc, s at uc, and min at uc. the fragment vp - d of n hrv strain was obtained by seminested pcr and specific primers vp f, and d inner and outer reverse primers (table ) , using takara lataq with gc buffer ii, for cycles of s at uc, s at uc, and min at uc. the terminal part of the whole genome was obtained by rapid amplification of cdna ends using / rapid amplification of the cdna kit, following the manufacturer's protocol (roche). to perform terminal race, ml of cdna buffer, ml dntps, . ml specific primer ( mm), . ml rna, ml control primer neo /rev ( . mm), ml control rna, ml rt enzyme, and . ml rnase inhibitor (roche) were mixed and incubated for uc for min, followed by inactivation at uc for min, and stored on ice. the product was purified using the qiagen pcr purification kit and eluted with ml deionized distilled water. a polya tail was added to the cdna, by mixing . ml dna with . ml reaction buffer, . ml ( mm) datp, and after incubation at uc for min, the reaction was chilled on ice for min. after addition of . ml terminal transferase, the reaction was incubated at uc for min, inactivated at uc for min, and kept on ice. nested pcr was performed by using the expend high fidelity pcr kit (roche). a mixture of . ml poly-da-tailed cdna, . ml oligodt-anchor primer . mm, . ml sp primers ( mm) (table ) , . ml control neo /rev primer ( . mm), . ml dntp, . ml enzyme, . ml buffer, and ml ddh o was incubated for cycles of s at uc, s at uc, and s at uc. to perform terminal race, the method was similar to normal two-step rt-pcr using d inner and outer f primers (table ) . sequence alignment, phylogenetic analyses and recombination analysis dna sequences used for p -p gene analysis were based on hrv- nt - and those used for vp /vp gene analysis were based on hrv- nt - . multiple sequences were aligned using clustal x [ ] . the multiple-sequence alignment was subjected to phylogenetic analyses using programs in the phylip package (v . ). bootstrap analysis was performed using seqboot, with a replicate number of . then, dnadist and neighbor were used to obtain distance matrices with the f parameter, and a transition/transversion ratio of . consensus trees were computed by consense, and then re-rooted with retree. the final tree was visualized and edited with mega version [ ] . recombination analysis was carried out by using recombination detection program v. . . manual bootscanning was performed by using the juke-cantor algorithm and the neighbor-joining method [ ] , with a window size of nt, a step size of nt and replicates. pairwise identities between sequences were determined with simplot software method [ ] ,with a window size of nt and a step size of nt. pairwise homology matrices were obtained by using clc combined workbench . software (clc bio, aarhus, denmark). the original p -vp sequences described in this study were deposited in genbank under accession nos. gq to gq . the vp -vp sequences were deposited under the nos gq to gq , and the p -p sequences under the nos gq to gq . the full length genomes sequences of n , n and n strains were deposited under the nos. gq , gq , gq , respectively. rhinovirus and the lower respiratory tract the common cold human rhinoviruses: the cold wars resume new developments in the epidemiology and clinical spectrum of rhinovirus infections association of respiratory picornaviruses with acute bronchiolitis in french infants respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis a novel group of rhinoviruses is associated with asthma hospitalizations respiratory illness caused by picornavirus infection: a review of clinical outcomes picornavirales, a proposed order of positive-sense single-stranded rna viruses with a pseudo-t = virion architecture phylogenetic analysis of human rhinovirus capsid protein vp and a protease coding sequences confirms shared genus-like relationships with human enteroviruses vp sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus assay for noncoding region analysis of all human rhinovirus prototype strains a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants sequencing and analyses of all known human rhinovirus genomes reveals structure and evolution global distribution of novel rhinovirus genotype pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany prior evidence of putative novel rhinovirus species noncoding region alone does not unequivocally determine genetic type of human rhinovirus strains distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) genome-wide diversity and selective pressure in the human rhinovirus frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections novel species of human rhinoviruses in acute otitis media human rhinovirus group c infection in children with lower respiratory tract infection novel human rhinoviruses and exacerbation of asthma in children genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees recombination in circulating enteroviruses improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing co-circulation and evolution of polioviruses and species c enteroviruses in a district of madagascar evidence of recombination among enteroviruses frequency and dynamics of recombination within different species of human enteroviruses genome analysis of circulating picornavirus reveals rhinovirus recombination and a new enterovirus serotype. xv european study group on the molecular biology of picornaviruses meeting enterovirus surveillance reveals proposed new serotypes and provides new insight into enterovirus -untranslated region evolution new respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality association of rhinovirus infection with increased disease severity in acute bronchiolitis the incidence of human bocavirus infection among children admitted to hospital in singapore human bocavirus: clinical significance and implications development of three multiplex rt-pcr assays for the detection of respiratory rna viruses detection of human bocavirus in hospitalised children clustal w and clustal x version . mega : molecular evolutionary genetics analysis (mega) software version . a modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints full-length human immunodeficiency virus type genomes from subtype cinfected seroconverters in india, with evidence of intersubtype recombination key: cord- -dez nzg authors: jartti, t.; kuusipalo, h.; vuorinen, t.; söderlund‐venermo, m.; allander, t.; waris, m.; hartiala, j.; ruuskanen, o. title: allergic sensitization is associated with rhinovirus‐, but not other virus‐, induced wheezing in children date: - - journal: pediatr allergy immunol doi: . /j. - . . .x sha: doc_id: cord_uid: dez nzg jartti t, kuusipalo h, vuorinen t, söderlund‐venermo m, allander t, waris m, hartiala j, ruuskanen o. allergic sensitization is associated with rhinovirus‐, but not other virus‐, induced wheezing in children. pediatr allergy immunol : : – . © john wiley & sons a/s background: data on the link between atopy and viral wheeze are limited. aim: to evaluate the association between ige sensitization and viral infection in wheezing children. methods: this is an observational study in hospitalized wheezing children (n = ; median age . ; interquartile range . , . ). eighteen respiratory viral infections were studied using all available methods. a specific immunoglobulin e (ige) sensitization for common food and aeroallergens and other atopy‐related variables including total ige, blood and nasal eosinophils, exhaled nitric oxide, eczema and atopic eczema, parental allergy and asthma, number of wheezing episodes, positive asthma predictive index or asthma and use of inhaled corticosteroid were correlated with specific viral etiology. results: atopy was closely associated with sole rhinovirus etiology (n = ) but not with sole respiratory syncytial virus, sole enterovirus, sole human bocavirus, sole other virus, mixed viral, or virus negative etiology. the number of sensitizations was particularly associated with sole rhinovirus etiology (odds ratio . ; % confidence interval . , . ; adjusted to age and sex), followed by aeroallergen sensitization (respectively; . ; . , . ), total ige level ( . ; . , . ), food allergen sensitization ( . ; . , . ), and nasal eosinophil count ( . ; . , . ). conclusions: according to our data, allergic sensitization is positively linked to rhinovirus‐, but not other virus‐, associated wheezing and calls attention for studies to test rhinovirus‐associated wheezing as a part of asthma risk indices. atopy, especially aeroallergen sensitization, is an important risk factor for chronic childhood asthma ( ) . aeroallergen sensitization, however, develops slowly, and it is rare during early life. thereby, it does not offer much help in the early identification of high-risk children ( , ) . on the contrary, respiratory viral infections are common already in young infants ( ) . the understanding of the link between atopy and respiratory viral infections is likely to give clinically highly relevant information that could help in designing early prevention and treatment strategies for asthma. therefore, in this observational analysis, we studied the link between allergic sensitization/several other atopy-related factors and respiratory viral infections (detected by all available modern molecular and conventional methods) in hospitalized wheezing children. we used a previously well-described cohort of hospitalized wheezing children ( ) . children aged months to years took part in a clinical trial (vinku-study) evaluating the efficacy of systemic corticosteroids for the treatment of virus-induced wheezing, which was the primary goal of the study ( , , ) . the study was carried out in the department of pediatrics, turku university hospital (turku, finland) from september, to may, (study breaks were june-july, and christmas week ). to this analysis, we included all children (n = ) with complete virology performed (fig. s ). the pre-defined inclusion criterion was acute wheezing necessitating hospitalization. pre-defined exclusion criteria included systemic corticosteroid treatment in the preceding weeks, chronic disease (other than atopyrelated disease), intensive care unit treatment, or previous participation in this study. the study was commenced only after obtaining written informed consent from the parents. the study protocol was approved by the ethics committee of the turku university hospital. atopy was defined as positive immunoglobulin (ig) e antibodies to any of the common allergens (cut-off level . ku/l for codfish, cowÕs milk, egg, peanut, soybean, wheat, cat, dog, horse, birch, mugwort, timothy, cladosporium herbarum, and dermatophagoides pteronyssinus; fluoro-enzyme immunoassay, cap feia, phadiatop combi Ò , phadia, uppsala, sweden). aeroallergen sensitization was defined as positive ige antibodies to any of the latter allergens. perennial aeroallergen sensitization was defined as positive ige antibodies to the dog, cat, or dermatophagoides pteronyssinus. birch, mugwort, timothy, and cladosporium herbarum were considered as seasonal aeroallergens. we slightly modified the asthma predictive index (api) according to the criteria for initiating daily long-term control therapy for asthma in children aged - as suggested by the heart, lung and blood institute of usa, i.e., ‡ wheezing episodes within the past year of which at least one confirmed by a physician, or prolonged symptoms lasting ‡ weeks and requiring symptomatic treatment > times per week, and in addition to either symptom criteria, ‡ major risk factors (physician diagnosed atopic eczema or parental asthma) or ‡ minor risk factors (allergic rhinitis, wheezing apart from colds or blood eosinophil count ‡ . · /l) originally introduced by castro-rodriguez et al. ( ) ( , ) . eczema (ever) was clinical diagnosis by a physician. it was defined as atopic eczema (ever) if specific sensitization (> . ku/l) was found. data on clinical food allergies or food challenges were not collected. on admission, a nasopharyngeal aspirate sample was obtained using a standardized procedure ( ) . blood samples were collected on admission and - weeks later. virus antigen detection and virus culture were analyzed using fresh samples by the department of virology, university of turku, and the samples obtained for polymerase chain reaction (pcr) assays were stored in ) °c before processing. blood counts and allergy tests were analyzed using fresh samples by the central laboratory or the turku university hospital. otherwise, sera were stored in ) °c before processing. virus culture was performed for adenovirus, influenza a and b viruses, parainfluenza virus (piv) types - , rsv, enteroviruses, hrv, and human metapneumovirus (hmpv) ( ) . viral antigens were detected for adenovirus, influenza a and b viruses, piv - , and rsv. levels of igg antibodies specific for adenovirus, influenza a and b viruses, piv / , rsv, and hbov were analyzed in paired serum samples, in addition to igm antibodies for enteroviruses and hbov ( , , , ) . pcr was used for the detection of hrv, enteroviruses, rsv, coronaviruses ( e, oc , nl and hku ), hmpv, hbov, influenza a and b viruses, adenovirus, piv - , and wu-and ki-polyoma viruses. moreover, formerly non-typable picornavirus and enterovirus samples were reanalyzed by rt-qpcr with improved identification of hrv-c strains ( ) . of available samples, / non-typable picornavirus samples and / ( %) enterovirus samples were hrv-c positive. hrv samples were not retested for hrv-c. non-typable picornavirus samples (rhino-enterovirus pcr positive samples that could not be typed by hybridization) were classified as rhinoviruses because all such samples, which were available for sequence analysis, showed rhinoviruses. exhaled nitric oxide was measured as previously described ( ) . the association analyses were performed by univariable and multivariable (adjusted to age and sex) logistic regression models. statistical significance was established at the level of p < . . we used sas/stat(r) software (version . . sp of the sas system for windows, sas institute inc., cary, nc, usa). of the enrolled children, had complete virology performed and were included in the analysis (fig. s ). the median age of the children was . years (range . , ; children were aged < ; children were aged - ; and children were aged > years). patient characteristics in sole virus, mixed virus, and virus-negative groups are partly shown in table (full details are shown in table s ). allergen-specific ige sensitization was closely associated with sole hrv etiology (n = ; fig. a ). log number of sensitizations were particularly associated with sole hrv etiology (odds ratio . ; adjusted to age and sex), followed by aeroallergen sensitization (respectively, . ), total ige level ( . ), food allergen sensitization ( . ), and nasal eosinophil count ( . ) (p < . for all, fig. b , table s ). as allergic sensitization is rare during early life, we further tested these associations in children aged < (n = ; children had sole hrv infection) and < years (n = ; children had sole hrv infection). in children aged < years, the associations were either noncomputable because of small sample sizes or nonsignificant (data not shown). in children aged < years, total ige level (odds ratio . ; % confidence interval . , . ; p = . ; after adjustments to age and gender, . ; . , . ; p = . ), any sensitization (respectively, . ; . , . ; p = . ), and food allergen sensitization ( . ; . , . ; p = . ) were positively associated with sole hrv etiology. the latter two associations did not persist after adjustments to age and gender. no other significant associations were found. levels of eosinophils, exhaled nitric oxide, and total ige and number of wheezing episodes were negatively associated with sole rsv etiology (n = ). log exhaled nitric oxide level was particularly associated with sole rsv etiology (odds ratio . ; adjusted to age and sex), followed by blood eosinophil count (respectively, . ), number of wheezing episodes ( . ), nasal eosinophil count ( . ), and log total ige level ( . ) (p < . for all, table s ). as allergic sensitization is rare during early life, we further tested these associations in children aged ‡ (n = ; children had sole rsv infection) and ‡ years (n = ; children had sole rsv infection). in children aged ‡ year, any sensitization (odds ratio . ; % confidence interval . , . ; p = . ; after adjustments to age and gender, respectively, . ; . , . ; p = . ) and nasal eosinophil count ( . ; . , . ; p = . ; after respective adjustments, . ; . , . ; p = . ) were negatively associated with sole rsv etiology. in children aged ‡ years, any sensitization was negatively associated with sole rsv etiology ( . ; . , . ; p = . ; statistical significance did not persist after respective adjustments). otherwise, the associations were either non-computable because of small sample sizes or non-significant (data not shown). eosinophil counts and use of inhaled corticosteroid at study entry were positively associated with sole enterovirus etiology (n = ). blood eosinophil count was particularly associated with sole enterovirus etiology (odds ratio . ; adjusted to age and sex), followed by nasal eosinophil count (respectively, . ) and use of inhaled corticosteroid at study entry ( . ) (p < . for all, table s ). in children aged < , there were sole enterovirus infections. the sizes of sole hbov group (n = ), sole other virus group (n = , which included: piv, n = ; hmpv = ; adenovirus, n = , influenza virus, n = , coronavirus, n = ), and virus negative group (n = ) were small. no significant associations were found between atopic characteristics and these groups (table s -s ) . atopic characteristics were not associated with mixed viral group (i.e., > viruses found per patient; patients; table s ). the following viruses were found in mixed viral infections: hrv (n = ), hbov (n = ), rsv (n = ), enteroviruses (n = ), piv (n = ), adenovirus (n = ), polyomaviruses (n = ), influenza virus (n = ), hmpv (n = ), and coronavirus (n = ). we show here that specific ige sensitization, particularly aeroallergen sensitization, is linked to hrv-induced acute wheezing, but not to acute wheezing induced by other viruses. this finding is in agreement with three previous studies that have connected hrv-associated wheezing to specific sensitization, nasal and systemic eosinophilia, and clinically diagnosed atopic eczema in separate reports ( , , ) . moreover, hyva¨rinen et al. ( ) ( ) found hrv infection to be dependent on atopy-related ) found hrv-associated wheezing during the third year of life to be independent risk factor for asthma at age , and hrv was eightfold stronger risk factor than aeroallergen sensitization. although rakes et al. ( ) ( ) found link between eosinophils and hrv infection that they also reported that hrv-negative wheezing children had more often specific sensitization than hrv-positive wheezing children. hrv diagnostics are based almost exclusively on pcr. considering the tremendous sensitivity of pcr-based hrv assay, does it detect true acute infections or likely remnants from past distant infections? hrv has been detected in up to % in asymptomatic children ( ) . it may represent a low-level infection without associated symptoms, although it is difficult to prove that a young child is totally asymptomatic. there is also evidence that positive hrv pcr finding may also represent the first sign of a developing clinical illness in about % to % of asymptomatic cases ( , ) . there are three other arguments suggesting that hrv pcr is likely to detect true infections, whether symptoms are present or not, and argue against the suggestion that viruses detected by pcr are likely to be residual nucleic acids left over from distant infections. first, the proportion of persistent or recurrent viral infections with same hrv strain is < % even in immunocompromised subjects ( , ) . second, although hrv detection rates are high in asymptomatic subject, hrv, any virus and mixed viral etiology has correlated with illness severity ( ) . third, hrv pcr findings have correlated with systemic cytokine responses ( ) . there are several mechanisms that could explain the link between atopy and hrv infection. first, atopic inflammation may increase the expression of the major hrv receptor, icam- (intercellular adhesion molecule ) ( ). the increased receptor levels are likely to lead to more severe hrv infections. in agreement, atopic individuals have shown more severe illnesses after experimental hrv inoculation, although there are also contradictory reports ( , ) . this mechanism does not apply to minor hrv group. second, t helper (th ) cell-polarized immune responses counteract th responses such as interferon-gamma, which belongs to nonspecific defense mechanisms against viral infections ( , ) . subjects with low interferon production have had greater susceptibility to hrv infections. third, a recent in vitro study showed that disrupted airway epithelium favoured hrv replication ( ) . interestingly, damaged airway epithelium opened the way to deeper cell layers where hrv replicated the most and also increased the number of its own receptor icam- . airway epithelium could be damaged by allergic inflammation, repeated respiratory infections, and/or by air pollution. fourth, high genetic diversity is a particular feature of hrv and makes it special from other viruses. today, - hrv serotypes and over different hrv genotypes have been found ( , ) . rhinoviruses elicit serotype-specific immunoresponses and are able to infect repeatedly with different strains. naturally, the high diversity and prevalence of hrv increase the odds that they could be associated with acute wheezing without causality. fifth, hrv-associated wheezing increases with age as does atopy. thus, the interaction between them is likely to be stronger by increasing age. therefore, we speculate that at young ages, when sensitization has not yet developed, the link may be more stronger between eosinophil levels and hrv infection. of note, the close association between enterovirus infection and eosinophilia may be explained by their phylogenetic similarity to hrv. the negative association between rsvinduced wheezing and certain atopy-related markers, such as nasal and blood eosinophils, exhaled nitric oxide, and total ige, is in agreement with three previous reports which have shown no association between rsv-induced bronchiolitis and sensitization status, eosinophil levels or presence of atopic eczema ( , , ) . moreover, in two long-term follow-up studies, a history of rsv-induced bronchiolitis or lower respiratory tract infection has been unrelated to atopy status at ages , , and - ( - ) . one study even reported a reduction in skin prick test positivity at the age of - in children with a history of hospitalization for rsv-induced lower respiratory tract infection when compared to matched controls ( ) . on the contrary, only one study has shown an increased association between rsv-induced bronchiolitis and sensitization to common allergens compared to matched controls at the age of ( ) . the negative/no association could be explained by several ways. first, rsv-affected children are typically young, median of months in our study, and sensitization rates and eosinophil levels are very low during infancy. second, rsv may not be truly linked to atopy in contrast to hrv, because we did not find positive link between sole rsv infection and atopic characteristics even in older children. third, eosinophils may be protective against rsv infection as shown by domachowske et al. ( ) ( ) . rsv and hrv were prominent viruses in the mixed virus group and probably had opposite effects within the group. the strength of the study was detailed virology and careful assessment of atopy status. there are also limitations. the power may not have been optimal for hbov etiology. the association between exhaled nitric oxide level and hbov showed an interesting positive tendency, and there were also wide confidence limits in the associations between sensitization variables and hbov infection. however, including also mixed viral infections (n = for sole/mixed hbov infections), atopy was not associated with hbov infection (data not shown). statistical power was also low for the other sole virus and virusnegative groups. one could argue that many other factors that are associated with either wheezing or susceptibility viral infections could confound our results. however, passive smoking (p > . ), day-care attendance (p > . ), number of siblings (p > . ), or presence of pets (p > . ) did not confound the hrv-or rsvrelated results (data not shown). we have no data on the duration of breast feeding. in conclusion, susceptibility to the common cold virus, hrv, associated wheezing is likely to be an early manifestation of biased immune functions and atopic airway inflammation. as atopy was not associated with none of the other viral, non-viral nor mixed viral infections, our observations together with previous long-term follow-up studies strengthen the role of hrv infection in wheezing children as an important tool for early identification of asthma-prone children and call attention for studies to include rhinovirus as a part of asthma risk indices ( , ) . additional supporting information may be found in the online version of this article: figure s . study flow chart. table s . the characteristics of hospitalized wheezing children with sole rhinovirus, sole respiratory syncytial virus (rsv), sole enterovirus, sole bocavirus, sole other virus, mixed viral and non-viral etiology. table s . the associations between atopic characteristics and sole rhinovirus infection (n = ) in hospitalized wheezing children. table s . the associations between atopic characteristics and sole respiratory syncytial virus infection (n = ) in hospitalized wheezing children. table s . the associations between atopic characteristics and sole enterovirus infection (n = ) in hospitalized wheezing children . table s . the associations between atopic characteristics and sole human bocavirus infection (n = ) in hospitalized wheezing children . table s . the associations between atopic characteristics and other sole virus infection than respiratory syncytial virus, rhinovirus, enterovirus, or bocavirus infection (n = ) in hospitalized wheezing children. table s . the associations between atopic characteristics and non-viral etiology (n = ) in hospitalized wheezing children. table s . the associations between atopic characteristics and mixed viral etiology (n = ) in hospitalized wheezing children. please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. human bocavirus and acute wheezing in children clinical assessment and improved diagnosis of bocavirus-induced wheezing in children rhinovirusinduced wheezing in infancy-the first sign of childhood asthma? rhinovirus illnesses during infancy predict subsequent childhood wheezing prednisolone reduces recurrent wheezing after a first wheezing episode associated with rhinovirus infection or eczema earlylife respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma wheezing rhinovirus illnesses in early life predict asthma development in high-risk children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis perennial allergen sensitisation early in life and chronic asthma in children: a birth cohort study evaluation of the efficacy of prednisolone in early wheezing induced by rhinovirus or respiratory syncytial virus serial viral infections in infants with recurrent respiratory illnesses respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children efficacy of prednisolone in children hospitalized for recurrent wheezing national heart, lung and blood institute. national asthma education and prevention program merkel cell polyomavirus dna in tumor-free tonsillar tissues and upper respiratory tract samples: implications for respiratory transmission and latency rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections teenage asthma after severe early childhood wheezing: an -year prospective follow-up persistence of rhinovirus and enterovirus rna after acute respiratory illness in children chronic rhinoviral infection in lung transplant recipients systemic t-helper and t-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study rhinovirus and the initiation of asthma host immune responses to rhinovirus: mechanisms in asthma basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants respiratory syncytial virus in early life and risk of wheeze and allergy by age years respiratory morbidity years after rsv infection in infancy hospitalization for rsv bronchiolitis before months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study association of an early respiratory syncytial virus infection and atopic allergy severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age evolution of antiviral activity in the ribonuclease a gene superfamily: evidence for a specific interaction between eosinophil-derived neurotoxin (edn/rnase ) and respiratory syncytial virus the clinical importance of rhinovirus-associated early wheezing inclusion of rhinovirus wheezing history in early life improves the sensitivity of the modified asthma predictive index (mapi) none. key: cord- -ysur sjq authors: nan title: respiratory nurses sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: ysur sjq nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- - y wdepc authors: traves, suzanne l; proud, david title: viral-associated exacerbations of asthma and copd date: - - journal: curr opin pharmacol doi: . /j.coph. . . sha: doc_id: cord_uid: y wdepc exacerbations of asthma and chronic obstructive pulmonary disease are major burdens on the healthcare system, and contribute significantly to the mortality and morbidity associated with these diseases. upper respiratory viral infections are associated with the majority of such disease exacerbations. the past few years have seen advances in the mechanisms by which viral infections induce pro-inflammatory chemokine production, and in our understanding of host antiviral and anti-inflammatory defence pathways that might regulate responses to infection. a more comprehensive understanding of the molecular basis of these processes could elucidate new therapeutic approaches to reduce the devastating impact that these exacerbations have on quality of life and healthcare costs. exacerbations of asthma and chronic obstructive pulmonary disease are major burdens on the healthcare system, and contribute significantly to the mortality and morbidity associated with these diseases. upper respiratory viral infections are associated with the majority of such disease exacerbations. the past few years have seen advances in the mechanisms by which viral infections induce pro-inflammatory chemokine production, and in our understanding of host antiviral and anti-inflammatory defence pathways that might regulate responses to infection. a more comprehensive understanding of the molecular basis of these processes could elucidate new therapeutic approaches to reduce the devastating impact that these exacerbations have on quality of life and healthcare costs. exacerbations of asthma and chronic obstructive pulmonary disease (copd) can be defined as 'a worsening of the patient's condition, beyond the day-to-day variability associated with the disease, that is sufficient enough to require a change in management, or to seek emergency medical intervention' [ , ] . exacerbations of both asthma and copd represent a major financial burden on the healthcare system as a result of costs associated with hospitalizations, increased medication usage, and days lost from work and school. in the case of asthma, exacerbations are responsible for % of the total healthcare costs, and for the deaths of americans each year [ ] . similarly in the case of copd, exacerbations account for % of healthcare costs, as well as being a substantial cause of hospitalizations [ ] . more importantly, recurrent exacerbations of copd result in a loss of lung function, thus hastening the progression of a currently fatal disease [ ] . in the current article, we discuss the evidence that common respiratory viruses are a major trigger factor for exacerbations of asthma and copd. we also review the current state of our knowledge on the mechanisms by which viruses might trigger such disease exacerbations, as well as the factors that could regulate susceptibility to viral exacerbations. finally, we consider the status of current therapies in the treatment of viral exacerbations of asthma and copd, and discuss potential novel approaches to treatment. the association between upper respiratory viral infections (uris) and exacerbations of asthma has been recognized for decades, but it was not until the development of rt-pcr methods for improved detection of viruses that the extent of this association became clear [ , ] . indeed, uris are the principal risk factor associated with asthma exacerbations [ , ] , and are associated with as many as - % of asthma exacerbations in children and adolescents [ , [ ] [ ] [ ] , and approximately - % of exacerbations in adults [ , ] . there is a clear temporal relationship between uris and asthma exacerbations in children. the peak of hospitalizations occurs in september, shortly after the return to school and at the peak time of year for human rhinovirus (hrv) infections [ , ] . consistent with this, hrv is associated with approximately % of viral-triggered exacerbations [ , , ] . other viral types associated with asthma exacerbations include influenza, coronaviruses, parainfluenza and respiratory syncytial virus. epidemiological evidence suggests that viruses may also interact with other causal factors linked to asthma exacerbations, such as allergens and pollution. studies of the interaction between experimental allergen exposure and experimental virus infection, however, have generated mixed results. in a murine model, influenza infection aids allergen sensitisation and enhances airway inflammation [ ] . in humans, however, chronic low-dose allergen provocations did not alter subsequent lower airway responses to hrv infection [ ] whereas, in the upper airways, acute allergen challenge delayed onset and shortened the duration of common colds. by , copd is predicted to become the third most common cause of death worldwide and the fifth leading cause of disability [ ] . exacerbations of copd occur more commonly in patients in the advanced gold ii or iii stages [ ] . (gold stands for the global initiative for chronic obstructive lung disease, and ranks disease in four stages: = at risk; stage i = mild copd; stage ii = moderate copd; stage iii = severe copd.) recent evidence has demonstrated that uris are a major trigger [ ] . in a recent study, % of severe exacerbations in patients with copd were associated with viral and/or bacterial infections, with viral infections accounting for % of these exacerbations [ ] . interestingly, viral-associated exacerbations of copd are more frequent, severe and have longer recovery times than those of non-viral origin [ ] . moreover, exacerbations associated with viral/bacterial co-infection also result in longer hospitalisation, and worse functional impairment for the patient [ ] . in patients with copd exacerbations requiring mechanical ventilation, a viral pathogen was detected in % of cases [ ] . in general, viral infections are responsible for approximately % of exacerbations of copd, with hrv being the dominant pathogen [ , ] . consistent with this, exacerbation frequency is associated with an increased frequency of acquiring the 'common cold' [ ] . moreover, experimental hrv infection in patients with gold stage ii copd resulted in symptoms and lung function changes representative of acute disease exacerbations [ ] . the specific mechanisms by which viruses invoke exacerbations of asthma and copd remain unclear. growing evidence, however, suggests direct infection of the lower respiratory tract, leading to a robust host inflammatory response, and an increase in bronchial hyperresponsiveness [ ] . because hrv is the major viral type associated with exacerbations, we focus on this virus as a prototype for the mechanisms by which viruses exert their effects. hrv infects both the upper and lower respiratory tracts, with the principal site of infection being the airway epithelial cell [ ] . although it has been reported that some strains of hrv can cause epithelial cell death in cultures grown at low density [ ] , the majority of studies found no overt cytotoxicity either in vitro or in vivo. epithelial cells are clearly the major site of hrv infection and sustain prolonged replication [ , ] . although hrv can bind to, and enter, a variety of other cell types in vitro, including fibroblasts, monocytes and macrophages [ , ] , the contribution of these individual cell types to pathogenesis in vivo is still unclear. hrv infection of cultured human airway epithelial cells results in production of several pro-inflammatory cytokines and chemokines, including interleukin (il)- , il- , il- , interferon (ifn)-inducible protein of kda (ip- ), regulated on activation normal t-cell expressed (rantes), granulocyte macrophage-colony stimulating factor and eotaxin [ ] [ ] [ ] . this profile of mediators could enhance airway inflammation via the recruitment and retention of a wide range of inflammatory cells ( figure ) that contribute to the pathogenesis of exacerbations [ ] . moreover, some of these chemokines have also been detected in airway secretions during viral infections [ ] . despite the potential for epithelial chemokines to recruit multiple cell types to the airways, experimental hrv infections and viral exacerbations of asthma and copd are dominantly associated with selective recruitment of neutrophils and lymphocytes [ ] . this implies that mechanisms must exist to limit the cell types recruited, but these mechanisms are not well understood. it has recently been reported that increased il- gene expression is observed during viral exacerbations of asthma, suggesting that immunoregulatory effects of il- include suppression of eosinophil influx [ ] . viral infections might also contribute to disease exacerbations by enhancing mucus production. hrv infection of epithelial cells has been shown to result in increased mrna expression for muc , muc , muc ac, muc b and muc . importantly, concentrations of muc ac and total mucin were also increased in supernatants and lysates from epithelial cells [ ] . although viral infection modulates epithelial cell function, the viral-induced signalling mechanisms involved are just beginning to be elucidated. induction of chemokines such as il- occurs early after hrv binding and does not require viral replication, but instead depends upon activation of both phosphatidylinositol -kinase and the p mitogen-activated protein kinase pathway [ ] . given that intercellular adhesion molecule- (icam- ), the receptor for the majority of hrv serotypes, has no inherent kinase activity or recognition motifs for receptorassociated kinases, it was unclear how viral binding initiated this signalling cascade. however, it has recently been shown that hrv binding to icam- leads to an association with the spleen tyrosine kinase syk. this association is mediated via the cytoskeletal linker protein ezrin, which binds both icam- and syk. formation of this complex leads to activation of syk, with subsequent downstream activation of the p mitogen-activated protein kinase pathway and increased expression of il- . thus, syk is an important signalling component in early virus responses [ ] . in contrast to the rapid generation of il- , other responses to viral infection, such as generation of rantes or ip- , do not occur until several hours after viral exposure and are absolutely dependent upon viral replication. this has led to intense investigation of the role of viral replication products, particularly double-stranded rna (dsrna), in cell responses. it is known that dsrna mimics several responses to viral infection and triggers host anti-viral responses. initially, dsrna was thought to be recognized exclusively by toll-like receptor (tlr) [ ] , but genetic deletion of tlr did not alter viral pathogenesis or host adaptive antiviral responses to several viruses [ ] . this apparent conundrum was resolved with the demonstration that two intracellular rna helicases -retinoic acid inducible gene (rig-i) [ , ] and melanomadifferentiation-associated gene (mda- ) [ ] -can also bind to dsrna. the helicase domain of these proteins binds dsrna, while a caspase activation and recruitment domain (card) permits binding of a downstream adaptor protein. this downstream mitochondrial-associated protein was identified independently by four groups and is known by the names cardif (card adapter inducing interferon-b), mavs (mitochondrial antiviral signalling), ips- (interferon-b promoter stimulator- ) and visa (virus-induced signalling adapter) [ ] . cardif binds to several proteins and induces both classical nuclear factor-kb (nf-kb) pathway activation and ikke/tankbinding kinase -mediated activation of interferon response factors [ ] . these pathways are also activated when dsrna binds to tlr (figure ) . controversy now exists regarding the relative roles of tlr , rig-i and mda- in viral recognition. it has been suggested that different viral types may preferentially utilize one of these three recognition proteins. to further complicate this picture, rig-i also recognizes single-stranded rna (ssrna) containing -phosphates. indeed, it has been reported that influenza a does not generate dsrna but, rather, activates rig-i via binding of genomic viral ssrna bearing -phosphates [ , ] . further studies are needed to clarify the preferential utilization of different intracellular recognition molecules by different viral types, and to determine if this also varies with cell type. mechanisms of virus-associated exacerbations of asthma and copd. viral infection of the epithelium results in the upregulation of icam- . pro-inflammatory mediators are also released that recruit inflammatory cells such as neutrophils and monocytes, which then differentiate into macrophages, lymphocytes and eosinophils. these inflammatory cells also release inflammatory mediators such as chemokines, cytokines, matrix metalloproteinases (mmps) and reactive oxygen species (ros), which perpetuate the inflammatory response culminating in an exacerbation. what determines susceptibility of a given individual to experience disease exacerbation upon viral infection is not clear but multiple factors are probably involved. those subjects whose underlying disease is well controlled are less likely to experience an acute viralmediated exacerbation. similarly, pre-existing specific immunity to a given pathogen will reduce the likelihood of that pathogen triggering an exacerbation. epithelial contributions to host innate antiviral immunity might also play a role. recent reports suggest that bronchial epithelial cells from asthmatic subjects show impaired production of both ifnb and type ifns [ , ] , and that this plays a role in the increased susceptibility of asthmatic subjects to lower airway disease. additional studies are needed to confirm these data and to put such defects in the context of why specific asthmatic subjects experience exacerbations. moreover, although type and type ifns contribute to host defence mainly via induction of numerous ifn-stimulated genes (isgs) that collectively limit virus replication and spread, there is a precedent for several viruses, including hrv, to induce isgs independently of ifn induction [ , ] . overview of the intracellular signalling pathway stimulated by rhinovirus. hrv binds to icam- on the surface of epithelial cells. the virus becomes internalised and, during replication, produces dsrna. dsrna can then bind tlr , which activates toll/il- -containing adaptor inducing interferon b (trif) and subsequently either interferon regulatory factor (irf) or nf-kb. dsrna can also bind either rig-i or mda- , resulting in the activation of irfor nf-kb-mediated pathways. it is thought that activation of irf and nf-kb stimulates the production of the anti-viral response. fadd, fas-associated death domain protein; ikk, inhibitor of nf-kb kinase family; ips- , interferon-b promoter stimulator ; mavs, mitochondrial antiviral signalling; rip- , kinase receptor interacting protein- ; tbk, tank-binding kinase; traf, tumor necrosis factor receptor-associated factor; visa, virus-induced signalling adaptor. nitric oxide (no) also appears to be an important component of the host antiviral response because it exerts direct antiviral activity against several viruses associated with exacerbations of asthma and copd, and also inhibits the viral-induced generation of several cytokines/chemokines from epithelial cells [ ] . viral infection of epithelial cells increases expression of inducible no synthase (inos) and, during in vivo hrv infections, epithelial inos induction correlates with levels of exhaled no. moreover, subjects with the highest levels of exhaled no cleared virus more rapidly and had fewer symptoms than those who exhaled lower levels [ ] . corticosteriods are crucial in the treatment of asthma [ ] and, when used alone or in combination with long-acting b-agonists or leukotriene receptor antagonists, they are known to improve asthma control and, thereby, reduce the number of exacerbations. acute asthma exacerbations tend to be treated with oxygen, inhaled short-acting b -adrenoceptor agonists, and intravenous or oral corticosteroids [ ] . although use of oral corticosteroids early in exacerbations can reduce subsequent relapse [ ] , there have been few studies looking specifically at exacerbations of known viral etiology. corticosteroids are ineffective in the treatment of hrvinduced colds and current evidence would suggest that they are of limited efficacy in viral-induced excerbations of asthma. asthmatics with prominent sputum neutrophilia, perhaps indicative of viral etiology, are poorly responsive to inhaled corticosteroids [ ] , and inhaled corticoisteroids did not significantly reduce lower airway inflammation induced by hrv infection of asthmatic subjects [ ] . moreover, administration of prednisolone to children hospitalized for viral-induced episodes of wheezy bronchiolitis did not reduce the duration of hospital stay [ ] . combination therapies of inhaled anticholinergic agents with short-acting b -adrenoceptor agonists have been reported to be more effective against exacerbations in school children [ ] ; furthermore, a leukotriene receptor antagonist decreased asthma exacerbations in -to -year-old patients with intermittent asthma [ ] . nedocromil sodium and inhaled corticosteroids might also be of limited benefit in asthma exacerbations in children [ ] . it must be noted, however, that most of these studies did not discriminate between exacerbations of viral and non-viral origin. although there is conflicting evidence over whether inhaled corticosteroids reduce exacerbations of copd, oral corticosteroids appear to hasten recovery from certain exacerbations [ ] . combined therapy with corticosteroids and long-acting b-adrenoceptor agonists has been shown to reduce the number of exacerbations in patients with copd, presumably by improving baseline control [ ] . again, however, there have been no studies specifi-cally examining the effects of corticosteroids, alone or in combination with long-acting b-adrenoceptor agonists, during copd exacerbations of known viral etiology. similarly, although antibiotic therapy is widely used in exacerbations of copd, their utility in virally triggered exacerbations is questionable. antiviral approaches appear to be a logical alternative to the treatment of viral exacerbations of asthma and copd, and influenza vaccine is clearly effective in preventing exacerbations triggered by this virus. vaccination approaches have not been successful for respiratory syncytial virus, however, and are not feasible for hrv, given the large number of viral serotypes. antiviral agents are available for influenza, and neuraminidase inhibitors have proven clinical efficacy in reducing the severity of symptoms during influenza infections. by contrast, antiviral approaches targeting hrv are still in development and have not yet been applied to viral exacerbations of asthma or copd. if the assumption that an over-exuberant host inflammatory response to viral infection plays a key role in disease exacerbation is valid, several potential therapeutic approaches can be suggested. the first would be to identify specific viral signaling pathways that would be targets for intervention. these could include, for example, specific early signaling pathways involving the spleen tyrosine kinase syk, or pathways triggered by viral interactions with the intracellular rna helicases. although targeting specific chemokines or chemokine receptors, such as cxcr or cxcr / , might also prove an attractive target, it remains to be determined which chemokine/chemokine-receptor systems are particularly important in disease pathogenesis. finally, enhancement of endogenous host antiviral pathways, or topical administration of drugs such as nitric oxide donors, could provide alternative approaches to reduce virally induced inflammation. there is an urgent need for additional therapeutic approaches to combat viral exacerbations of asthma and copd. although the past few years have seen a significant increase in our understanding of how viruses cause exacerbations, much remains to be learned. the complex signalling pathways triggered upon viral infection are not completely understood but, once delineated, could provide novel therapeutic targets. in addition, better understanding of host innate antiviral mechanisms could provide an alternative therapeutic approach if such pathways can be stimulated. pathophysiology of exacerbations of chronic obstructive pulmonary disease chronic obstructive pulmonary disease. : the aetiology of exacerbations of chronic obstructive pulmonary disease role of viral infections in asthma and chronic obstructive pulmonary disease how viral infections cause exacerbation of airway diseases allergens, viruses, and asthma exacerbations the role of rhinovirus in asthma exacerbations persistence of rhinovirus rna after asthma exacerbation in children study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing the september epidemic of asthma hospitalization: school children as disease vectors the relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking unravelling synergistic immune interactions between respiratory virus infections and allergic airway inflammation bronchial matrix and inflammation respond to inhaled steroids despite ongoing allergen exposure in asthma the global burden of disease molecular mechanisms of respiratory virus-induced asthma and copd exacerbations and pneumonia role of viruses in exacerbations of chronic obstructive pulmonary disease infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation epidemiological relationships between the common cold and exacerbation frequency in copd an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells proud d: human airway epithelial cells produce ip- (cxcl ) in vitro and in vivo upon rhinovirus infection rhinovirus induces airway epithelial gene expression through double-stranded rna and ifn-dependent pathways human rhinovirus induces robust ip- release by monocytic cells, which is independent of viral replication but linked to type i interferon receptor ligation and stat activation host defense function of the airway epithelium in health and disease: clinical background interleukin- gene expression in acute virus-induced asthma mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells phosphatidylinositol -kinase is required for rhinovirus-induced airway epithelial cell interleukin- expression syk is downstream of intercellular adhesion molecule- and mediates human rhinovirus activation of p mapk in airway epithelial cells the authors demonstrate for the first time a molecular mechanism linking rhinovirus binding to icam- and downstream mediator production. they highlight the role of syk and ezrin as important signalling molecules following rhinovirus infection of epithelial cells recognition of double-stranded rna and activation of nf-kappab by toll-like receptor does toll-like receptor play a biological role in virus infections? the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses differential roles of mda and rig-i helicases in the recognition of rna viruses using knockout mice for rig- and mda- , the authors show that these mice are more susceptible to viral infection, highlighting the role of these proteins in the anti-viral respsonse. they also demonstrate for the first time that rig-i and mda- bind different viruses, and that synthetic dsrna poly(i:c) mediates its effects via mda- card games between virus and host get a new player this short article brings together four independent studies which discovered the same card-containing protein (cardif/mavs/ips- /visa) that mediates the effects of rig-i and mda- . it outlines the major findings from the four groups, and highlights that possible signalling pathways of dsrna produced by the virus -triphosphate rna is the ligand for rig-i reis e sousa: rig-i-mediated antiviral responses to single-stranded rna bearing phosphates these two articles highlight for the first time that rig- can also bind ssrna, and that the -triphosphate is crucial for rig- activation. these articles are important, as it was previously thought that rig- could only bind dsrna asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient type iii interferon-lambda production in asthma exacerbations they highlight deficiencies in the immune response to infection in asthma. they found that the production of ifnb and ifnl was severely impaired in epithelial cells and alveolar macrophages during asthma exacerbations, thus highlighting these compounds as potential therapies. . noyce rs nitric oxide and the common cold role of nasal nitric oxide in the resolution of experimental rhinovirus infection systemic corticosteroid therapy for acute asthma exacerbations comparison of asthma treatment given in addition to inhaled corticosteroids on airway inflammation and responsiveness rhinovirus-induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during experimental infection evaluation of the efficacy of prednisolone in early wheezing induced by rhinovirus or respiratory syncytial virus polos p: montelukast reduces asthma exacerbations in -to -year-old children with intermittent asthma limitations of maintenance therapy for viral respiratory infection-induced asthma dr traves acknowledges altana canada for postdoctoral fellowship support. dr proud is the recipient of a canada research chair in inflammatory airway diseases and thanks the canadian institutes of health research for support of our research. key: cord- -vjh z l authors: storch, gregory a title: respiratory viruses in babies: important insights from down under date: - - journal: j infect dis doi: . /infdis/jix sha: doc_id: cord_uid: vjh z l nan for those of us interested in respiratory viruses, there is a lot to like about the study by sarna et al that appears in this issue of the journal of infectious diseases [ ] . the study analyzes data from a birth cohort established as part of the observational research in childhood infectious diseases (orchid) project, based in brisbane, australia [ ] . impressive study attributes include large size, community base, enrollment from birth, scheduled frequent longitudinal sampling with or without illness, high percentage of specimen acquisition rate, even enrollment of subjects throughout the year to account for virus seasonality, and testing of samples with an extensive panel of real-time polymerase chain reaction (pcr) assays. participating babies had anterior nasal and "dirty nappy" swabs obtained at birth and weekly thereafter by parents trained by research staff. parents also kept daily symptom diaries listing predefined symptoms. the present manuscript reports only on the respiratory swabs. this massive undertaking yielded analyzable data on samples from participants, corresponding to individual virus-specific pcr queries! this intensive prospective study of respiratory viruses in infants finds that human rhinovirus (hrv) is by far the most frequent virus detected in the infant respiratory tract. this single-stranded, positive-sense rna virus is increasingly recognized as an important human pathogen, with a strong relationship to childhood asthma [ ] . significantly, sarna et al found that hrv was detected in an impressive . % of all samples tested, accounting for . % of all virus-positive swabs. by years of age, % of participating infants had experienced hrv-c, % hrv-a, and % hrv-b. although impressive, this large proportional load of hrv should not be surprising, as it has also been shown in previous community-based studies using molecular tests, including an earlier study by these authors [ ] and several others [ ] [ ] [ ] [ ] . similarly, laboratories using multiplex respiratory virus panels that include sensitive rhinovirus detection capability usually find rhinovirus/enterovirus to be the most common positive result. the us food and drug administration (fda)-cleared multiplex respiratory panels do not distinguish between rhinoviruses and enteroviruses, but apart from outbreak situations, such as occurred in with enterovirus d- [ ] , most of the positive results from rhinovirus/enterovirus assays reflect hrv infection. it has become clear that in relation to respiratory viruses, hrv is the elephant in the room. in comparison to the universal occurrence of hrv, infections with other respiratory viruses were less frequent. rsv and parainfluenza viruses were each detected in % of subjects, influenza a in %, influenza b in %, human metapneumovirus in %, human coronaviruses in %, adenovirus in %, human polyomaviruses in %, and human bocavirus in %. the % occurrence of rsv is surprisingly low, as common understanding is that almost all infants experience rsv within the first years of life [ ] . possible explanations for the discrepancy include inadequate sample collection (samples were collected by parents), seasonal variation in the occurrence of rsv, and complete reliance on molecular assays. in some studies, serology has indicated some infections are not detected by pcr [ ] . the authors point out the difference between their estimate and that of the houston family study [ ] , which is a basis for the concept of universal rsv infection early in life and which relied heavily on serology [ ] . however, they also cite a number of more recent studies that support their finding of less than universal infection. these results suggest that we may need to fine-tune our understanding of the frequency of rsv infection early in life. the focus of the sarna et al study was first respiratory virus infections, and the frequency of first hrv infections in young infants is noteworthy. of the infants followed, % experienced a first infection with hrv by months of age, compared to . % for rsv, and . %- . % for the other respiratory viruses. influenza a and b infections were infrequent, with only . % and . % experiencing first influenza a and b, respectively, by months of age. the curves mapping virus occurrence by age show that all viruses other than hrv were relatively unusual in the first months of life, with upward inflection points evident at approximately months of age, consistent with diminishing maternal immunity. notably, the hrv occurrence curve is different, with rapid increase through the first months and no obvious inflection point. the shape of this curve suggests that maternal immunity is not preventing hrv infections, although it is possible that it is mitigating clinical manifestations. this finding has important implications for hrv control strategies, and calls for mechanistic studies to further define the differences between the behavior of hrv and other respiratory viruses. with mounting undeniable evidence for the frequent occurrence of hrv infection in the first years of life, it is important to assess the impact of these infections, and sarna et al provide relevant data. based on symptom diaries kept by parents, virus-positive episodes were characterized as symptomatic or asymptomatic and, if symptomatic, as involving the upper or lower respiratory tract. if we direct attention to the episodes in which only a single virus was detected (data shown in supplementary table of the sarna et al manuscript) , we see that first hrv detections were less likely than first infections with the other rna viruses to correspond to symptomatic episodes ( % compared to %- % for the other rna viruses), but were comparably likely compared to the dna viruses for which %- % of first detection episodes corresponded to symptomatic infections. likewise, only % of first hrv infections were classified as lower respiratory infections, compared with %- % for the other rna viruses and %- % for the dna viruses. only % of the first hrv detections were associated with a medical visit, compared with %- % for the other rna viruses and %- % for the dna viruses. it should be noted that direct comparison of severity of hrv infections to the other respiratory viruses is complicated by the fact that a much higher proportion of first episode hrv infections occurred in infants < months of age, and data are not provided (and may not have been available because of the infrequency of infection with the non-hrv respiratory viruses during that age period) that allow us to ascertain whether an age effect is present. nevertheless, it is clear that most of the first hrv infections were mild, in spite of the fact that they were occurring predominantly in infants < months of age, an age when the immune system is not fully developed and some viral infections can disseminate. however, as the authors correctly point out, the mildness of the clinical illness associated with first hrv detections may not be the whole story. they cite a recent study by wolsk et al [ ] showing that hrv infection in the first weeks of life, even if asymptomatic, may program immune memory with an exaggerated t-helper mucosal immune response and impaired antiviral responses. the implication is that hrv infection in young infants, even if asymptomatic, might promote the development of asthma later in life. this interaction may be viewed as hrv serving as an educator (or miseducator) of the immune system by virtue of early-in-life virus-host interactions. this concept fits well with an emerging view that one of the key functions of the infant's microbiome is education of the immune system [ ] . the orchid study has made an important contribution by directing our attention to the extremely frequent interaction between hrv and the immature immune system, the implications of which clearly merit further study. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. potential conflicts of interest. author confirmed no potential conflicts. the author have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. timing of first respiratory virus detection in infants: a community-based birth cohort study observational research in childhood infectious diseases (orchid): a dynamic birth cohort study understanding the association of human rhinovirus with asthma community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study swiss paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study community surveillance of respiratory viruses among families in the utah better identification of • editorial commentary germs-longitudinal viral epidemiology (big-love) study etiology of acute respiratory infections in infants: a prospective birth cohort study the emergence of enterovirus d report of the committee on infectious diseases serology enhances molecular diagnosis of respiratory virus infections other than influenza in children and adults hospitalized with community-acquired pneumonia risk of primary infection and reinfection with respiratory syncytial virus picornavirus-induced airway mucosa immune profile in asymptomatic neonates host-microbiota interactions and adaptive immunity key: cord- -ta k uyz authors: etemadi, mohammad reza; othman, norlijah; savolainen-kopra, carita; sekawi, zamberi; wahab, noraabd; sann, lye munn title: biodiversity and clinico-demographic characteristics of human rhinoviruses from hospitalized children with acute lower respiratory tract infections in malaysia() date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: ta k uyz background: there is accumulating evidence that human rhinovirus (hrv) causes acute lower respiratory tract infections (alrti). recently, hrv-c was identified as a new species of hrv, but its spectrum of clinical disease is not well understood. objectives: we investigated the molecular epidemiology, demographic and clinical characteristics of hrvs among hospitalized children with alris. study design: one hundred and sixty-five nasopharangeal aspirates taken from children < years hospitalized with alrtis in serdang hospital, malaysia, were subject to reverse transcriptase-pcr for hrv. phylogenetic analysis on vp /vp and ′-ncr regions was used to further characterize hrv. other respiratory viruses were also investigated using semi-nested multiplex rt-pcr assay. clinical parameters were analyzed between hrv, rsv and ifv-a mono-infections and between hrv species. results: hrv was detected in ( %) patients for both single ( samples) and multiple ( samples) infections, . % ( / ) represents hrv-a strains while the remaining hrv-c. strain p was the first reported representative of hrv . the majority of the single hrv cases were in the second half of infancy; hrv-c occurred among older children compared with hrv-a. hrv children were admitted significantly earlier and less febrile than rsv and ifv-a infection. hrv-c infected children were more likely to have rhonchi and vomiting as compared to hrv-a. pneumonia was the most common discharge diagnosis followed by bronchiolitis and post-viral wheeze in hrv patients. conclusion: our study showed high prevalence of hrvs and detection of hrv-c among hospitalized children with alrtis in malaysia. analysis of clinical parameters suggested specific features associated with hrvs infections and specific hrv groups. sequences in genomic databases revealing greater association of hrvs with serious alrtis such as deadly pneumonia [ ] [ ] [ ] [ ] [ ] . the presence of the new hrv-c strain in severe respiratory disease has further instilled research interest in the clinical impact, molecular biology and epidemiology of hrvs. as research of hrv is limited [ ] , especially in asian developing countries, this study aims to examine the molecular epidemiology, the demographic characteristics and clinical features including the newly discovered hrv-c species, among hospitalized children less than years of age with alrti in malaysia. a study was conducted among children more than one-monthold and less than years of age with the diagnosis of alrti to the pediatric wards at hospital serdang, selangor, malaysia, from june to december , , after obtaining approval from both the medical research and ethics committee (mrec) of the ministry of health malaysia and university putra malaysia. selection of subjects was based on predetermined inclusion and exclusion criteria. children who met the final diagnosis for alrti had at least of the following: fever, tachypnoea, cough, auscultation findings indicative of lower respiratory disease (including rhonchi, crackles, or bronchial breath sounds) and/or chest retraction supported with chest radiographic changes, if available, were included and if nasopharangeal specimens were collected within h of admission. children who had one of the followings were excluded from the study: congenital or acquired immunosuppressive conditions and patients with conditions posing a potential hazard in obtaining the nasopharyngeal samples. a standardized study protocol was developed to record demographic and medical history of the patients, clinical features including symptoms and signs, outcome of the illness and hospital course, and laboratory and radiological findings. at the end of the hospitalization, the children's charts were reviewed by four of the co-investigators and the clinical diagnosis was determined by the principal investigator. the clinical diagnosis was categorized into pneumonia, bronchiolitis, and post-viral wheeze. during the first h of admission, nasopharyngeal aspirates (npa) were taken from each patient. viral genome was extracted using magmax viral rna isolation kit (applied biosystems, ca, usa). detection of hrv was performed via reverse transcription on rna extracts using revertaid h minus first strand cdna synthesis kit (fermentase, usa). pcr was performed for all the samples using primers that amplified a fragment encompassing the vp /vp region and the hyper-variable region in the -ncr of human rhinoviruses [ ] . both strands of pcr products were sequenced using abi xl dna analyzer (applied biosystems). specimens were also being tested for respiratory syncytial virus (rsv), human metapneumovirus (hmpv), influenza virus (ifv) type a and b, parainfluenzavirus - (piv - ), coronaviruses (hcov) oc and e [ ] , human bocavirus (hbov) and human adenovirus (hadv) using pcr [ , ] . raw sequence data were analyzed using vector nti suite (invitrogen corp., carlsbad, ca, usa), assembled using contig express and aligned using alignx software. multiple sequence alignments were made utilizing mega software [ ] with default parameters followed by manual editing. phylogenetic tree was estimated with mega using neighbor-joining method [ ] with maximum composite likelihood model with bootstrap replicates [ ] . the nucleotide and deduced amino acid sequences of the vp /vp regions were compared with those of hrv-a, hrv-b and hrv-c strains using reference sequences available in the genbank using blast (basic local alignment search toolhttp://blast.ncbi.nlm.nih.gov/). the sequences generated in this study were submitted to genbank under accession numbers hm to hm . demographic and clinical parameters were compared between hrv, rsv, and ifv-a mono infections and also among hrv species. pearson chi-square or fisher's exact test were used for categorical variables. the student's independent sample ttest or non-parametric mann-whitney u test, anova tests or kruskal-wallis test were applied for continuous variables where it's applicable. all analyses were performed using spss version . . p-values of < . were considered statistically significant. a total of children < years of age who fulfilled the inclusion criteria were enrolled in the study. rsv ( / , . %) was found to be the main single virus detected from the study population, followed by hrv ( / , . %) and ifv-a ( / , . %). multiple hrv infections were found in samples ( . %), including in dual and in triple infections as shown in table . of these, dual infection of hrv and rsv ( / , . %) was the most prevalent multiple infection recorded. in total, out of ( . %) samples were found infected with hrv. out of hrv single infections, ( . %) were male. the mean age of hrv patients was . ranging from . to . months. they were older than children infected with rsv and younger than ifv-a patients (mean age of and . months respectively). the mean age of hrv-c infections was significantly higher than hrv-a infections ( . vs. . months). the majority of the hrv-a infections were found in children of - months of age; while the peak age of patients hospitalized with hrv-c was - months. the clinical features of children infected with hrv, rsv and ifv-a are shown in table . hrv infected patients were admitted earlier compared to rsv and influenza; children with hrv presented to the hospital after a mean duration of . days (ranged - days) as compared with hrv ( . days, p = < . ) and ifv-a ( . days, p = . ). in terms of respiratory features, there were no distinctive differences among the three infections. however, fever occurred less often in hrv infections than in rsv and ifv-a. temperature equal or above • c was presented in % of hrv as compared with % of rsv and % of ifv-a infections. majority of the hrv patients were hospitalized for shorter duration and received less antibiotic, particularly in comparison to rsv. disease severity characterized by need of oxygen, admission to intensive care unit (only one case of hrv) and prolonged hospital stay did not differ among the studied virus groups. diarrhea was less common in hrv single infections as compared with ifv-a ( . % vs. %, respectively, p = . ). leukocytosis occurred significantly more frequently in hrv single infections than in rsv (p = . ). differential count showed neutrophilic predominance in hrv compared with rsv patients (p = . ). pneumonia was the most common discharge diagnosis among hrv patients followed by bronchiolitis and post-viral wheeze. vomiting occurred almost two times more with hrv-c infection (p = . ), as shown in table . hrv-c patients were more likely to present with rhonchi in comparison to hrv-a ( % vs. %, respectively, p = . ). the study also showed that hrv-c patients tended to have a higher neutrophil count (p = . ) while lymphocytosis was associated with hrv-a (p = . ). post-viral wheeze was more common with hrv-c infections. disease severity characterized by need of oxygen, admission to intensive care unit (only one case of hrv) and prolonged hospital stay did not differ among the studied species. hrv single infections were subjected to sequencing as the study aimed at looking into different variables of the single infections. thirty-three of hrv single samples were retrieved in sequencing. additionally three hrv samples co-infected with other viruses were also sequenced. all genetically typed hrv strains ( ). we found that ( %) of the samples were clustered in the separate clad distinct from hrv-a and hrv-b, along with representative strains of hrv-c (fig. ) . hrv-b strains were not among the typed strains. the nucleotide similarities to closest prototype strain in hrv-a, varied from . % to . %, while the closest genetic relatives were observed with a variation range of . % to . %. the respective hrv-c strain similarities varied from . % to . %. genetic analysis used for typing revealed four human enterovirus (hev) strains in species hev-b, hev-c and hev-d. however, it should be noted here that the vp /vp region used for genetic typing of hrv strains does not unequivocally allow typing of hev strains due to greater sequence similarity between strains within species. a significant burden of hrv infection, was found in out of patients ( %), and this concurs with previous studies with a rate varying from % to % in a hospitalized pediatric patient [ , [ ] [ ] [ ] [ ] [ ] . this contradicts with the traditional notion that hrv is only associated with upper respiratory tract infections. earlier studies indicated that the clinical value of positive pcr is somewhat disputable among patients with alrti [ , , , ] as asymptomatic hrv infections is known to occur in - % of individuals. several subsequent clinical studies of small numbers of selected patients showed that hrv can replicate in lower respiratory tract. this finding concluded that detection of hrv especially in children less than years does not simply represent asymptomatic infection but is related to true infection [ , ] . in addition, the ability of hrv to infect lower respiratory airways and to induce cytotoxicity to bronchial epithelium has been shown experimentally among immunocompetent individuals [ ] . in this study the most prevalent combination was found between hrv and rsv. the combination of hrv and rsv as a main double infection has been reported by others [ , , , ] . high incidence of hrv coupled with rsv infection could be explained by the substantial overlapping of monthly distribution observed for these viruses during the study period [ ] . the relatively recent application of molecular study of pcr based methods for hrv has identified several novel hrvs, including hrv-c. our study showed that hrv-b was absent while hrv-a predominates from hrv-c. this finding seems to reinforce that hrv-b is a minor species worldwide; however it concurs with most findings by other investigators as hrv-a was the most prevalent compared to the other species. this may reflect apparent seasonality or yearly variation of circulating hrv. on the other hand, it may be associated with hrv-b of a milder form of disease presentation and consequent of lesser rate of hospitalization. consistent with other findings, our study showed that hrv infected children are older than those with rsv infections and younger than those with ifv-a infections [ , ] . the results support the importance of hrv infections among infants with acute respiratory infections [ , ] especially in its second half. hrv-a infections were more likely to occur during infancy as compared with hrv-c, further corroborating the findings that hrv-a infections are less prevalent in older age group as compared with hrv-c [ , ] . few studies have described the clinical characteristics of hrv alri in infants and young children. our study revealed that hrv infected children were hospitalized earlier in the course of their disease and were less febrile on presentation as compared to rsv and ifv-a infections. they were also more likely to be discharged earlier and tend to receive lesser antibiotics as compared to the other two infections, especially to rsv. hrv is known to have a shorter incubation period and hence presented earlier as compared with hrv or occasionally with ifv-a especially with h n infection. this could be explained further in terms of the cytopathology on the respiratory epithelial cells as hrv is the least invasive with minimal cell damage and secondary bacterial infection is far less a complication compared to rsv and ifv. as a result, patients were subjected to lesser antibiotics and stayed shorter in the hospital as seen in this study. however, neutrophilia as documented in the present study may not reflect secondary bacterial infection in hrv. several studies in the past, acknowledged the increase infection in both peripheral and airway neutrophils as a risk to development of asthmatic exacerbations. on the other hand, ifa-a infection tended to be associated with diarrhea, as the infection usually resulted in table clinical characteristics of hrv-a and hrv-c single infections. a more systemic involvement with direct invasion of the gastrointestine by the virus [ ] . this study further distinguishes certain clinical features of the two species of hrv in alrti. although in general, the clinical presentations were comparable for both hrv-a and c, rhonchi and vomiting were more common in hrv-c infected children as compared to hrv-a. in our study, the presence of rhonchi was associated with alrti rather than asthma, as only one had asthma as an underlying disease. as shown in previous studies, this finding further supports the role of hrv-c among patients with febrile wheeze in alrti. a large clinical cohort study indicated that this group of children has a preexisting predisposition to asthma in early childhood. on the other hand, vomiting has not been reported in other studies as a prominent presentation in hrv-c. vomiting with coughing is common in children (post-tussive vomiting) and it could be related to the wheezing episodes in our study and indicate a more severe form of infection in hrv-c as compared to hrv-a. likewise, no characteristic laboratory finding has been associated previously with any specific hrv species; the modest increase in lymphocytes in hrv-a and neutrophils in hrv-c in our study could be coincidental and probably of no clinical importance. consistent with the study by jin [ ] , there was no significant difference in terms of disease severity for both species of hrv. on the other hand, miller [ ] found that hrv-c patients were more likely to require supplemental oxygen than hrv-a. the variations could be partly attributed to bacterial co-infections, viral load and type of the studied population. therefore, these data must be interpreted with some caution. several different types were detected including outbreak-like clusters, e.g. hrv . phylogenetic analysis confirmed global prevalence of hrv-c strains. this study showed the large variation of concomitantly circulating hrv strains as reported previously [ ] . due to increased sequence typing efforts during recent years, most of the hrv strains of this study were shown to have very close genetic relatives circulating in other geographical areas. in this study, hrv has been implicated as a cause of hospitalization in children with alrti in our locality. the dominant presence of both hrv-a and c concurs with global epidemiologic studies in other parts of the world. there were also some differences between the demographic variables, clinical manifestations and laboratory findings of hrv, rsv and ifv-a infections and within the hrv species. however, corroboration of the clinical significance and its pathogenesis will require larger numbers of subjects and should include a control group with no respiratory infection. the study could not be generalized as only it involved small numbers and in-patients are involved. a prospective longitudinal multicenter population based studies, utilizing quantitative pcr methods, are needed to better explore and understand the role of hrv in alrtis. this work was partially supported by grants from the ministry of science, technology and innovation malaysia (grant number ). the isolation of a new virus associated with respiratory clinical disease in humans masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life a birth cohort study etiology of community-acquired pneumonia in hospitalized children etiology of communityacquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines rhinoviruses are a major cause of wheezing and hospitalization in children less than years of age human rhinovirus c associated with wheezing in hospitalised children in the middle east genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus development of three multiplex rt-pcr assays for the detection of respiratory rna viruses molecular typing of human adenoviruses by pcr and sequencing of a partial region of the hexon gene cloning of a human parvovirus by molecular screening of respiratory tract samples molecular evolutionary genetics analysis (mega) software version . the neighbor-joining method: a new method for reconstructing phylogenetic trees an empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis detection of viruses identified recently in children with acute wheezing clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup association of rhinovirus infection with increased disease severity in acute bronchiolitis rhinovirus infections in children: a retrospective and prospective hospital-based study rhinovirus-associated hospitalizations in young children clinical effects of rhinovirus infections respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children mixed respiratory virus infections the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis a novel group of rhinoviruses is associated with asthma hospitalizations high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease influenza virus a infections presenting with febrile convulsions and gastrointestinal symptoms in young children prevalence and clinical characterization of a newly identified human rhinovirus c species in children with acute respiratory tract infections phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons we would like to extend our appreciation to the director of health for allowing us to carry out the above study. we are also grateful to all the doctors and nurses of the pediatric department, hospital serdang for extending their kind assistance in facilitating the study. we thank dr. sharifah aishah, the radiologist for her expertise in the interpretation and reporting of the radiographs. the authors involved in this study have no conflicts of interest to declare. approval was from both the medical research and ethics committee (mrec) of the ministry of health malaysia and university putra malaysia. informed consent was obtained from cares of the patients. key: cord- -vnn b authors: lee, wai-ming; kiesner, christin; pappas, tressa; lee, iris; grindle, kris; jartti, tuomas; jakiela, bogdan; lemanske, robert f.; shult, peter a.; gern, james e. title: a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vnn b background: human rhinoviruses (hrvs) are the most prevalent human pathogens, and consist of serotypes that are classified into groups a and b according to sequence variations. hrv infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. defining the role of specific strains in various hrv illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using serotype-specific antisera, is insensitive and laborious. methods and findings: to directly type hrvs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a -bp variable sequence in the ' noncoding region with homologous sequences of the known serotypes. nasal samples from infants were first tested with a multiplex pcr assay for respiratory viruses, and hrv was the most common virus found ( of samples). typing was completed for samples and hrvs were identified. surprisingly, ( . %) hrvs did not match any of the known serotypes and had – % nucleotide divergence from the nearest reference hrvs. of these novel viruses, strains ( hrvs) segregated from hrva, hrvb and human enterovirus into a distinct genetic group (“c”). none of these new strains could be cultured in traditional cell lines. conclusions: by molecular analysis, over % of hrv detected in sick infants were previously unrecognized strains, including strains that may represent a new hrv group. these findings indicate that the number of hrv strains is considerably larger than the serotypes identified with traditional diagnostic techniques, and provide evidence of a new hrv group. human rhinoviruses (hrvs), members of picornavirus family, are small nonenveloped viruses with a -base mrna positive sense rna genome [ ] . the first hrv was discovered in [ , ] , and by , serotypes ( a and b to ) were identified using susceptible cell cultures and specific antisera [ , , ] . multiple epidemiologic studies of serotype circulation conducted between [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] showed that . % of field isolates could be identified with the serotype-specific antisera prepared before , and many serotypes identified earlier were still circulating [ , , ] . these results suggested hrv serotypes are stable and do not undergo influenza virus-like antigenic drift [ ] . hrvs are the most prevalent human respiratory pathogens [ , , , , ] . annually, hrvs are responsible for . % of all acute upper respiratory illness (common colds), the most frequent human illness. hrv infections occur year round worldwide and are epidemic in early fall and late spring in the temperate regions. hrv infections cause a wide range of clinical outcomes including asymptomatic infections, [ , , , , ] upper respiratory illnesses, and in children, asthmatics, and other susceptible populations, lower respiratory symptoms. [ , , , , , ] . defining the role of specific strains in various hrv illnesses has been difficult because traditional serology requires the isolation of hrv in susceptible cell cultures and neutralization tests against all serotype-specific antisera [ ] . this traditional serological method is insensitive, labor intensive and cumbersome [ ] . more sensitive and faster molecular methods have been developed for serotyping enteroviruses, which are closely related to hrv [ ] . in addition, molecular typing methods have been used to identify the links between illnesses and specific strains of pathogens such as dengue viruses, influenza viruses, human papillomaviruses, hepatitis c viruses, and hiv [ , ] . molecular typing involves pcr amplification of a portion of the target viral genome, sequencing and phylogenetic analyses. in this report, we analyzed clinical specimens from sick infants with a new molecular method, and identified new hrv strains including that constitute a new hrv group. the length of the p -p sequences (region between primer sites p and p in figure ) varied only slightly between the established serotypes, ranging from to bases. the maximum pairwise nucleotide divergence (%) between all serotypes in this region was % ( figure ). this result was similar to the maximum pairwise divergence of vp sequences ( %) and slightly lower than that of vp sequences ( %) [ , ] . furthermore, . % of all the serotype pairs had . % pairwise nucleotide divergence. the maximum pairwise divergences (%) of p -p sequences among hrva and hrvb viruses were % and %, respectively. these results demonstrated the potential utility of this region for differentiating hrv serotypes. p -p sequences of hrv serotypes clustered into previously defined genetic groups: hrva and hrvb phylogenetic tree reconstruction confirmed that the p -p sequences clustered into genetic groups, a and b, (figure ). the p -p phylogenetic distribution of the serotypes into group was identical to that of published trees based on vp and vp -vp sequences [ , , ] , with the same serotypes in the hrva group and serotypes in hrvb group ( figure ). the topology of the p -p tree was similar to that of the vp and vp -vp trees [ , , ] . these results agreed with previous reports that the nucleotide phylogenies of hrvs are consistent across the whole genome, from 'ncr through polyprotein to 'ncr [ ] . accurate typing of clinical isolates by phylogenetic tree reconstruction of p -p sequences to test whether p -p sequences were suitable for hrv serotype identification, we compared the typing results of culturable clinical isolates obtained by p -p sequences with those by nim- a sequences of vp . the degenerate primers ev and ev for pcr amplification of nim- a region were not sensitive enough for direct detection of small amount of hrv in original clinical samples (data not shown), and high titer infected cell lysates of cultured isolates were needed to produce enough pcr product for cloning and sequencing. the length of nim- a and p -p sequences ranged - bases and - bases, respectively. based on phylogenetic tree reconstruction of nim- a sequences, the clinical isolates were assigned to different serotypes with highly significant bootstrap values ( - %, table ). identical assignment results were obtained with p -p tree, although a number of assignments (hrv b, hrv , hrv and one of the hrv ) had low but still significant bootstrap values ( %, %, % and %, respectively). interestingly, the nucleotide divergence between the clinical isolates and the respective reference strain was lower at the p -p region (mean . %, range - %) than at the nim- a region (mean . %, range - %) ( table ) . this result agreed with previous reports that nucleotide sequences were more conserved at the ncrs than the coding region due to the preservation of conserved rna structure elements within the ncr [ , ] . nasal lavage samples of illnesses from infants with frequent respiratory illnesses were analyzed by respiratory multicode assay [ ] . hrv was detected in samples ( %) ( table ) . other viruses detected were enterovirus, rsv, adenovirus, coronavirus, influenza a virus, metapneumovirus and parainfluenza virus. among the hrv-positive samples, had only hrv and had coinfection with at least one other respiratory virus. the identity of hrv in of the samples was determined by molecular typing. of the samples that were not typed, had no sample left and did not yield p -p fragments by semi-nested pcr. a total of hrvs were identified in samples. only samples contained different hrvs, indicating a low rate of infection with more than one strain. of the hrvs, serotypes were assigned to hrvs by phylogenetic tree reconstruction ( table ). the assignments of hrvs were strongly supported by highly significant bootstrap values (. %). one assignment (hrv ) had a low but still significant bootstrap value ( %). the p -p sequences of these hrvs had - % identity with the respective reference serotypes. three sequences clustered with hrv but had poor bootstrap values (, %) although they had % identity with hrv reference sequences. this was probably because these hrvs also matched well with hrv , which has % identity with hrv in the p -p region. these hrvs and p ) and variable region between p and p (p -p in red) at the 'ncr and the pcr fragments used in this study. p , p and p are located at bases - , - and - , respectively in hrv genome. pcr fragment a (about bps) was used to determine the 'ncr sequences of all hrv serotypes. it was amplified using pan-hrv pcr forward primer p - , which anneals to conserved region p , and a serotype-specific reverse primer annealed to the ' end of vp gene (between base# and ). pcr fragment b (about bps) was generated with pan-hrv pcr forward primer p - and reverse primer p - . pcr fragment c (about bps) was generated with forward primer p - and an equimolar mixture of reverse primers p - , p - and p - . the variable sequences of p -p were used for the molecular typing assay. doi: . /journal.pone. .g were grouped into hrv serotypes; ( serotypes) were hrva and ( serotypes) were hrvb viruses ( table ) . the p -p sequences of hrvs did not cluster with the homologous sequences of any of the serotypes in phylogenetic tree reconstructions (figure ). these hrvs clustered into new unique strains with high degree of nucleotide divergence from the respective nearest reference serotypes (mean . %, range - %) and between strains (mean . %, range - %) ( table ). different new hrvs of the same strain had a high degree of identity among themselves (mean . %, range - %, table ). seventeen of the new strains clustered with group a hrv ( figure ) and had - % pairwise nucleotide divergence from the nearest reference serotype (table ). among them, strains (w , w , w , w and w ) clustered in a distant branch near serotypes , and ; strains (w , w , w , w , w , w and w ) in distant branch near serotypes , and ; strains w , w , w and w formed a new branch, and strain w was the lone member of a new branch. moreover, strains (w , w , w , w , w , w , w , w and w ) separated from both hrva and hrvb ( figure ) and had - % pairwise divergence from the nearest reference serotype (table ). we propose that they represent a new hrv genetic group (hrvc). none of the new strains clustered with hrvb viruses. interestingly, none of the samples containing the new hrv strains produced cpe in standard wi- or mrc- cell cultures used for the detection and isolation of hrv (data not shown). hevs are closely related to hrvs [ ] , and comprise . distinct serotypes that include polioviruses, coxsackieviruses a and b, echoviruses and the newer numbered evs. hevs are classified into groups: poliovirus and human enterovirus a-d (hev-a-d), according to both biological and molecular properties. like hrvs, some hevs are upper respiratory pathogens. to determine the relationship of hrvc to hevs, phylogenetic tree reconstruction was performed with the p -p sequences of hrvc strains and respective sequences of all known hev (n = ). the results indicate that hrvc viruses are distinct from hev: the pairwise divergence of p -p sequences between a hrvc strain and the respective nearest hev ranged from % to % ( figure ). in this report we demonstrate that the pool of circulating hrv strains is significantly larger than the collection of known serotypes. less than half of the hrv detected in these young infants corresponded to previously recognized serotypes (table ) , and are previously unrecognized hrvs belonging to new strains (table ). moreover, of the strains form a new genetic group "c" that is distinct from the previously defined groups hrva and hrvb and the human enteroviruses ( figure ). the remaining new strains cluster into existing distant or new branches in hrva group ( figure ) . interestingly, none of the new viruses grew in standard tissue culture, which may explain why these viruses were previously undetected. these new hrv strains were detected with a sensitive molecular method to type hrv directly from the original clinical specimens. this new assay had key components: sensitive pan-hrv primers and semi-nested pcr to amplify p -p region from cdna prepared from original clinical specimens, a sequence database of -bp p -p region of 'ncr of all hrv serotypes to serve as standard references for hrv identification, and phylogenetic tree reconstruction of the new p -p sequences and the homologous reference sequences. phylogenetic tree reconstruction has been shown to be more accurate than the other sequence analysis methods for molecular typing of enteroviruses [ ] . interestingly, the 'ncr is not suitable for typing of enterovirus. for example, the 'ncr sequence of coxackieviruses do not correlate with serotype and vp sequence due to frequent recombination at the 'ncr [ , ] . in contrast, hrv maintains consistent phylogeny across its genome and has limited recombination [ ] , and this characteristic enables 'ncr sequence (p -p ) to be used for strain identification. for enteroviruses, vp sequencing is commonly used for molecular serotyping because this region contains major antigenic sites that correlate well with serotype [ ] . however, vp sequences of hrv are less conserved, and degenerate primer pairs such as ev and ev [ ] that target this region were relatively insensitive for pcr amplification. the established hrv serotypes (hrv a to hrv ) were discovered and designated between and . they were isolated using susceptible wi- cell cultures and then defined with serotype-specific antisera [ , , ] . multiple epidemiologic studies of serotype circulation conducted between - showed that . % of the field isolates could be identified with the serotype-specific antisera (hrv a through hrv ) prepared before and many serotypes identified earlier were still circulating [ , ] [ , ] . these results suggested that almost all hrv serotypes had already been identified and new serotypes were not evolving [ , ] . in fact, only one possible new serotype, hrv-hanks, was reported in the next years ( to ), and it was subsequently shown to be hrv by careful sequence analysis and neutralization testing [ ] . recently, studies utilizing molecular techniques instead of culture-based diagnostics have provided evidence of additional strains of hrv [ , ] . for example, lamson and colleagues identified new hrvs in new york state (ny) by vp sequencing. they concluded that these hrvs represented a new genetic clade because they clustered in a branch at the root of the hrva phylogenetic tree [ ] . in addition, mcerlean and colleagues obtained the complete genome sequence of a new hrv strain, hrv-qpm, in queensland, australia. they showed by phylogenetic analysis that hrv-qpm was a new member of hrva and belonged to a new genetic sub-lineage of hrva, hrv-a , and the new ny hrvs also belonged to hrv-a [ ] . to compare the identities of these newly reported hrvs with our new strains, we performed phylogenetic tree reconstruction using p -p sequences of our new strains, hrv-qpm, and the established serotypes; and then a similar analysis using vp sequences of hrv-qpm, ny hrvs, and the established serotypes. the p -p phylogenetic tree (not shown) revealed that hrv-qpm and one of our new hrva viruses, w , were the same strain and thus supported mcerlean's conclusion that hrv-qpm was a group a virus. the vp tree (not shown) confirmed mcerlean's finding that hrv-qpm and the ny hrvs belonged to the same cluster within hrva group. therefore the ny hrvs and hrv-qpm were related to our new hrva strains. in contrast, our hrvc strains form a distinct group separated from both hrva and hrvb (figure ) . analysis of 'ncr of all hrv serotypes reveals that there are both highly conserved sequences (e.g. p , p and p primer sites, figure ) and also variable sequences between p and p . the -bp p -p region had up to % pairwise nucleotide divergence between serotypes, similar to that of vp ( %) and vp ( %). moreover, . % of all the p -p pairs from distinct serotypes had . % pairwise nucleotide divergence. despite this inter-serotype variability, the p -p sequence of 'ncr was significantly more conserved between clinical isolates and the corresponding reference prototype strain (mean divergence . %) compared to the nim- a coding sequences of vp (mean divergence . %, table ). these data suggest that 'ncr sequences may be under greater selective constraint, and it is known that this region contains rna structure elements that are critical for viral replication and translation [ ] . in summary, more than half ( %) of the hrvs detected in these young infants were new hrv strains, and many of them clustered into a distinct genetic group c. these findings indicate that the number of hrv strains has been markedly underestimated by traditional viral culture and serotyping techniques, and also raise additional questions. first, how widespread are these this tree was generated as described in figure . none of the new strains clustered with hrvb viruses. seventeen new strains (blue) belonged to hrva group, and strains (red) cluster into a new group (''c'') that is separate from groups a and b. doi: . /journal.pone. .g putative ''group c'' hrv? our study population consisted of a group of infants who experienced frequent illnesses, and additional studies are needed to define the spectrum of hrv infections in unselected populations, and in subjects of other age groups. secondly, is the biology of these novel strains similar to that of other hrv? the inability to culture these viruses suggests that receptor utilization or other growth requirements are distinct. the development of molecular assays for the detection and analysis of respiratory viruses provide important tools for new epidemiologic and mechanistic studies to address these questions. a more complete understanding of the spectrum of respiratory viruses and their biology is essential for efforts directed at prevention and treatment of these common and clinically significant illnesses. this study was approved by the human subject committee of the school of medicine and public health, university of wisconsin -madison. written informed consent was obtained from the parents. for determining the standard reference sequences, infected cell lysates of prototype strains of hrv serotypes were obtained from dr. fred hayden of u. virginia, charlottesville (serotype a, b, to , to , to and hanks) and atcc (hrv and hrv ). hrv was excluded from the reference database because it has been reclassified as a human enterovirus [ , ] . clinical samples were obtained from two sources. first, samples were obtained from infants ages - year participating in a prospective birth cohort study (childhood origins of asthma) in wisconsin to determine the role of viral and host factors in the pathogenesis of asthma [ ] . of the children who completed the first year of the study, infants had frequent ($ ) moderate to severe respiratory illnesses, and samples from were available for further study. nasal lavage specimens were collected from illnesses between spring of and spring of . second, for validation of our molecular typing assay, infected cell lysates of additional hrv clinical isolates were obtained from wisconsin state laboratory of hygiene (wslh). these clinical isolates were recovered from nasal lavages of infants in and using wi cell culture and identified by the characteristic cytopathic effect and acid lability of hrv [ ] . pairwise sequence alignment, multiple sequence alignment, % identity calculation, distance (divergence) calculation, phylogenetic tree reconstruction and bootstrap analysis were performed using software clustal . . [ , ] . in a typical analysis, the input sequences were first processed to produce a multiple alignment and matrixes of identity and distances (divergence) between all sequence pairs. the distance matrix was used by the neighbor joining method to produce an unrooted phylogenetic tree with branch length proportional to the divergence of the sequences. the confidence of the clustering of sequences was evaluated by bootstrapping ( replicates). bootstrap values of . ( %) indicate the highly significant clustering, whereas values , ( %) indicate that the clustering is not statistically significant. the phylogenetic tree with bootstrap values was visualized using software njplot. preparation of cdna from nasal specimens cdna preparation was performed as described elsewhere [ ] . briefly, nasal fluid ( ml) was mixed with extraction carriers (glycogen and glycoblue) and ml of trizol ls (invitrogen ), vortexed for minutes, supplied with ml of chloroform, vortexed again for minutes and then microfuged for minutes. the supernatant aqueous phase (, ml) was mixed with ml isopropanol and this mixture was incubated at room temperature for hr. the rna precipitant was pelleted by microfugation for minutes, washed once with % ethanol, airdried and then dissolved in ml water. to make cdna, ml of rna solution was mixed with ml of reaction solution containing promega amv-reverse transcriptase, amv-rt buffer, random primers, rnasin and dntps and then incubated at uc for minutes, uc for minutes, uc for minutes, and uc for minutes. rma is a new high-throughput, multiplex pcr-microsphere flow cytometry assay system for comprehensive detection of common respiratory viruses including rhinoviruses (hrv), enteroviruses, respiratory syncytial viruses (rsv), parainfluenza viruses, influenza viruses, metapneumoviruses, adenoviruses and coronaviruses. details of the rma assay have been previously described [ ] . the output signal is expressed as mfi (median fluorescence intensity), and samples with an average signal . standard deviations of average negative control signals (typically to mfi) are regarded as positive. the rma is capable of distinguishing closely related hrv and enteroviruses [ ] . selection of the target region to identify a genomic region suitable for molecular typing of hrv, we analyzed all published hrv sequences. these included complete genome sequences of serotypes ( b, , , , , , and ) [ , , , , , , , ] [ , , ] , d (rna polymerase) sequences of serotypes [ ] and partial 'ncr sequences of serotypes [ , , ] . careful alignment analysis of these sequences showed that only the 'ncr region had highly conserved sequences (p , p and p regions, figure ) that could be used for making pan-hrv primers, and a long variable sequence (p -p , figure ) suitable for serotype differentiation. however, 'ncr sequences were available for only a fraction of serotypes. cloning and sequencing of the 'ncr of reference hrv serotypes to establish a reference sequence database for molecular typing, we cloned and sequenced the 'ncr of all hrv serotypes. detailed viral rna preparation, rt-pcr, cloning and sequencing procedure have been described elsewhere [ ] . briefly, total nucleic acids were prepared from ml of infected cell lysate by phenol extraction and ethanol precipitation. rt (reverse transcription)-pcr was performed in a rt-pcr mix (invitrogen - ) using the following conditions: min at uc, min at uc, cycles of ( sec at uc, sec at uc, sec at uc) and min at uc. the pcr primer pairs were forward primer p - (caagcacttctgtywcccc) for all serotypes and a serotype-specific reverse primer. primer p - was designed within the conserved p region (figure ). the reverse primer was selected for each of the serotypes within a region corresponding to bases - of hrv near the ' end of vp gene ( figure ) according to published sequences [ ] . the pcr product, a dna fragment of about bp covering % of the 'ncr, complete vp gene and about bases of vp gene (pcr fragment a of figure ), was isolated by agarose gel electrophoresis. after the agarose was removed by phenol extraction, pcr fragments were treated with kinase, ligated to a stui-linearized plamsid vector pmj , and then transformed into e. coli. three plasmids with pcr fragment inserted were isolated for each serotype, amplified and purified. each viral dna fragment was completely sequenced (automated dna sequencing facility, u. wisconsin). the serotype identity of each sequence was verified by matching of its vp /vp sequence to the respective published sequence [ ] . semi-nested pcr amplification of p -p region from original clinical specimens primer p - , which amplified the 'ncr of all serotypes, was chosen as the forward primer. for reverse primers, multiple candidates were designed within highly conserved p and p regions (figure ). primers p - (acgg-acacccaaagtag), p - (ttagccacattcaggggc), p - (ttagccacattcaggagcc) and p - (ttagcc-gcattcagggg) were selected based on efficient hrv sequence amplification. for the first pcr, . ml of leftover cdna from the rma assay was added to a tube containing ml platinum pcr supermix hf (invitrogen - ), ml of forward primer p - ( mm) and ml of reverse primer p - ( mm). the reaction started with min at uc, followed by a 'touchdown' cycle (the steps were uc for s, uc down to uc ( uc intervals) for s, and then uc for s. there were two cycles for each annealing temperature down to uc followed by cycles at uc, and then a final min at uc. this reaction produces a pcr product of bp (figure , fragment b) . for the second pcr, ml of the first pcr product was transferred to a new pcr tube containing ml platinum pcr supermix hf, ml of forward primer p - ( mm) and ml of each reverse primer p - ( mm), p - ( mm) and p - ( mm). the reaction conditions are min at uc, cycles of ( sec at uc, sec at uc, sec at uc) and min at uc. the final product is a bp dna fragment ( figure , fragment c) . this semi-nested pcr protocol requires only copies of cdna template per sample to produce sufficient product for cloning and does not produce nonspecific product from original clinical specimens (data not shown). fragment c was then purified, cloned, and plasmids containing fragment c were isolated and sequenced for each sample. for samples, different p -p sequences were found, indicating the presence of more than one serotype/strain, so additional plasmids were analyzed. sequencing of the vp and 'ncr of hrv clinical isolates to determine whether the p -p variable sequences within the 'ncr were suitable for the differentiation and identification of hrv, we compared the typing results by p -p sequences with those of nim- a sequences of vp . nim- a is a dominant antigenic site of hrv first identified for hrv [ ] , and the homologous sequences of this region correlate well with serotype of hrvs and enteroviruses [ , ] . viral rna was isolated from virus-infected culture supernatant of hrv clinical isolates with qiaamp viral rna mini kit (qiagen ) as described [ ] . to obtain the vp sequences, viral rna was first amplified using primer pair ev and ev as described [ ] with modified rt-pcr conditions: min at uc, min at uc, min at uc, cycles of ( sec at uc, sec at uc, sec at uc) and min at uc. the pcr product (, bp) was isolated by agarose gel electrophoresis and then sequenced [ ] . to obtain the 'ncr sequences, viral rna was amplified by rt-pcr using p - and p - primers, cloned and sequenced. assignment of serotypes and new strain a phylogenetic tree with bootstrap values and a matrix of % pairwise nucleotide divergence (distance %) were generated for each new sequence (nim- a or p -p ) and homologous reference sequences using clustal with default alignment parameters, which were selected after testing a range of parameters. next, a new isolate or detection was assigned the serotype to which it clustered with in the phylogenetic tree with a significant bootstrap value (. %) [ ] . in contrast, if the p -p sequence of a new isolate or detection did not cluster with one of the serotypes in the phylogenetic tree, and had . % pairwise nucleotide divergence from the nearest reference serotype, it was designated as a new strain (prefixed with w). the threshold pairwise divergence value ( %) for assigning a new strain was determined after considering the pairwise divergence values between the known serotypes (figure ) , and the pairwise divergence values between reference serotypes and clinical isolates of the same serotype (table , last column). two types of errors could be made in evaluating two sequences: a) deciding they were the same when they were actually different, and b) deciding they were different when they were actually the same. the probability of these errors was determined empirically using the data shown in figure and table , respectively. according to figure , %, %, %, %, and % of the p -p pairs of all serotypes had . %, . %, . %, . % and . % pairwise nucleotide divergence, respectively. as shown in table (last column), the pairwise nucleotide divergence of all p -p pairs between clinical isolates and the respective reference serotypes was consistently # %. therefore, a threshold of % is associated with probabilities of , % and , % for errors a and b, respectively. the original p -p sequences described in this report have been deposited in the genbank sequence database under accession no. eu to eu . picornavirus structure and multiplication a cytopathogenic agent isolated from naval recruits with mild respiratory illnesses the isolation of a new virus associated with respiratory clinical disease in humans a collaborative report: rhinoviruses-extension of the numbering system rhinoviruses: a numbering system a collaborative report: rhinoviruses-extension of the numbering system from to rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes the common cold rhinoviruses: important respiratory pathogens epidemiology of viral respiratory infections clinical virology a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection the september epidemic of asthma exacerbations in children: a search for etiology use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms predominance of rhinovirus in the nose of symptomatic and asymptomatic infants picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season respiratory illness caused by picornavirus infection: a review of clinical outcomes picornavirus infections: a primer for the practitioner rhinovirus: more than just a common cold virus rhinovirus respiratory infections and asthma rhinovirus infections: more than a common cold rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing typing of human enteroviruses by partial sequencing of vp the application of molecular phylogenetics to the analysis of viral genome diversity and evolution molecular epidemiology and evolution of emerging infectious diseases phylogenetic analysis of human rhinovirus capsid protein vp and a protease coding sequences confirms shared genus-like relationships with human enteroviruses genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus vp sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds genome-wide diversity and selective pressure in the human rhinovirus conserved rna secondary structures in picornaviridae genomes highthroughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses molecular identification of enterovirus by analyzing a partial vp genomic region with different methods molecular evolution of the human enteroviruses: correlation of serotype with vp sequence and application to picornavirus classification genotypic variation in coxsackievirus b isolates from three different outbreaks in the united states masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis enterovirus is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses human rhinovirus and enterovirus represent a unique serotype with rhinovirus and enterovirus features rhinovirus illnesses during infancy predict subsequent childhood wheezing relationships among specific viral pathogens, virus-induced interleukin- , and respiratory symptoms in infancy multiple sequence alignment with the clustal series of programs the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools amino acid changes in proteins b and a mediate rhinovirus type growth in mouse cells molecular cloning and complete sequence determination of rna genome of human rhinovirus type the complete nucleotide sequence of a common cold virus: human rhinovirus the nucleotide sequence of human rhinovirus b: molecular relationships within the rhinovirus genus role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome complete sequence of the rna genome of human rhinovirus , a clinically useful common cold virus belonging to the icam- receptor group evolutionary relationships within the human rhinovirus genus: comparison of serotypes , , and human rhinovirus : complete nucleotide sequence and proteolytic processing signals in the capsid protein region sequence analysis of human rhinoviruses in the rna-dependent rna polymerase coding region reveals large within-species variation improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the ' noncoding regions of additional rhinovirus strains improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay amplicon sequencing and improved detection of human rhinovirus in respiratory samples sensitive, seminested pcr amplification of vp sequences for direct identification of all enterovirus serotypes from original clinical specimens comparison of classic and molecular approaches for the identification of untypeable enteroviruses we thank dr. fred hayden (u. virginia, charlottesville) for generously providing infected cell lysates of prototype hrvstrains. we also thank dr. key: cord- -daz vokz authors: devereux, graham; matsui, elizabeth c.; burney, peter g.j. title: epidemiology of asthma and allergic airway diseases date: - - journal: middleton's allergy doi: . /b - - - - . - sha: doc_id: cord_uid: daz vokz nan epidemiology is the study of the distribution of disease in populations. it is essential for assessing the spread and burden of disease. it is the appropriate method for understanding the cause and pathogenesis of disease. research into allergy has had a long history with many changes in direction, and the language that has been developed to describe what has been found has changed over time. this can lead to confusion. in this chapter, we use the term sensitization to indicate the production of immunoglobulin e (ige) antibodies in response to allergens. we use the term allergy to refer to the presence of one or more diseases associated with ige sensitization, the most common of which are asthma, eczema, and rhinitis. the term atopy was originally introduced to account for the observation that the main allergic diseases occurred in the same families and appeared to have a common origin. however, it is often used synonymously with the term allergy. test standards. good tests should possess reliability and validity. a test is reliable if it always gives the same answer when applied under similar circumstances. validity implies that the result of the test coincides well with the true condition of the person being tested. validity has two components: sensitivity, which is the ability of the test to identify an existing condition, and specificity, which is the ability to identify as normal people who are free of the condition. measuring the validity of a test for a condition that is poorly defined, such as asthma, is a problem because it presupposes a gold standard test with which the proposed test can be compared. although validity in an absolute sense may always be contested, what is as important in epidemiologic studies is standardization, meaning that the test is identical wherever and by whomever it is administered. validity is essential to the measurement of absolute prevalence, but in many epidemiologic studies, we are as interested in relative prevalence, such as relative prevalence between age groups, countries, or districts, or differences between people exposed to various environmental or genetic risks. standardization is essential for this, and considerable effort has been made to provide standardized measures, particularly for international studies. tests of sensitization. sensitization can be assessed directly by determining the presence of specific ige to allergens in serum. in many places, mites, grass, and cat allergens are among the most common allergens, and most sensitized individuals can be identified by testing for relatively few allergens. , some test kits can identify a mixture of several allergens. in the past, they have been used to test for the occurrence of sensitization, and this may be cost-effective, but it leaves unclear which allergens are » epidemiology is the study of the distribution of disease and, by extension, its causes and consequences, mostly in general populations. » the rates of allergic sensitization and allergic diseases have been increasing, although the increase in prevalence of allergic diseases has slowed among children. » allergic disease is less common in rural parts of low-income countries, although allergic sensitization can be common in these areas. » there has been very little success in explaining the increased prevalence of allergic disease, although it has been linked to urbanization. the great changes observed in prevalence and distribution strongly suggest a major role for the environment. » factors that initiate allergy and allergic diseases should be differentiated from factors that exacerbate them after they have been established. » allergies are affected by environmental factors, including diet; exposure to a normal, diverse microflora; infections; exposure to air pollutants; and occupational exposures. » allergy is not associated with higher mortality rates or loss of lung function, but asthma is associated with both. » outcomes for asthma can be considerably improved by good management. test of whether someone had asthma. what had been provided was, in his view, no more than a description. second, he pointed out that most diseases were concepts rather than "things" and that their definitions were therefore bound to be contested. since then, there have been many attempts to define asthma (table - ) , although most have paid little attention to the issues raised by this led to more complicated descriptions but not to any greater clarity. some have introduced additional assumptions about mechanisms and causes. despite these strictures, asthma has been an enduring and trusted concept clinically, but a separate question remains about how the condition can be identified in epidemiologic studies. there are effectively three broad methods of identifying asthma in surveys: questionnaires asking about diagnosed asthma, questions about the symptoms of asthma, and physiologic tests of airway responsiveness. questions asking whether someone has asthma, often qualified by asking whether a doctor has ever confirmed the diagnosis, are common. they are regarded as highly specific, meaning that there are few people who answer this question in the affirmative but do not have asthma, but there are many people who may be defined as asthmatic who deny that they have the condition. the worst characteristic of these questions is the lack of standardization. the answers to the questions depend on local medical practice and the terms used by health professionals when talking to patients. variations in the use of the term asthma likely have influenced estimates of time trends and observed differences in mortality between countries. over the past years, the prevalence of people with asthma has increased markedly, and there has been much debate about whether this can be explained by differences in the way the term has been used. this possibility is supported by the encouragement given to pediatricians, particularly from the s onward, to diagnose all wheezy children as asthmatic because this would encourage the use of medication and was shown to enhance the quality of life of children regardless of the exact diagnosis. in the s, kelson and heller sent scenarios of patients who had died to a representative group of physicians signing death certificates in several european countries. one scenario (box - ) described a person who had some symptoms of asthma but many of the features of chronic obstructive lung disease. figure - shows the relationship between the proportion of the physicians in each country ascribing this death to asthma and the national mortality rate for asthma. there is a strong suggestion that the way doctors in each country view such marginal cases may be influencing the national mortality data. whether this is still the case is uncertain. since then, there has been a major increase in international consensus documents. asking about symptoms rather than diagnosed disease avoids some of these problems, and efforts have been made to find suitable questionnaires and to standardize them across countries. the most commonly used questionnaire for children is that developed for the international study of asthma and allergies in childhood (isaac). for adults, the questionnaire developed for the international union against tuberculosis and lung disease (iuatld) , was subsequently adapted for use in the european community respiratory health survey (ecrhs) and was further adapted for the world health survey. responsible for symptoms. microchip technology and the development of recombinant and purified allergens have enabled testing for several allergens simultaneously and allowed more precise identification of the relevant allergens. the technology remains expensive and is not widely used in epidemiologic studies. an alternative method of identifying sensitized individuals is to undertake skin-prick tests. they do not require a laboratory and do not involve taking blood. the technique involves introducing a small amount of allergen under the outer layers of the skin using a needle or lancet and reading the size of the wheal that appears in the minutes after the test is applied. this is compared with the wheal produced by a control solution (usually the diluent in which the allergens have been dissolved) and with a positive (usually histamine) control that tests whether the skin is able to respond to the release of mediators that the allergen induces. skin tests have more operatordependent variation than serologic tests, because they are influenced in part by the technique of the technician, but they typically are cheap and provide an immediate answer, which can be more satisfactory for the patient or participant. the criterion for a positive test result varies according to the purpose of testing. using any test greater than the diluent control is more repeatable and less prone to observational error and reflects well the presence of allergen-specific ige. however, in a clinical context, small wheals are rarely associated with allergic disease that can be ascribed to that allergen, and in a clinical context, wheals less than mm in diameter usually are discounted as irrelevant. defining the prevalence of sensitization in a population depends to some extent on which allergens are tested. in western europe and the united states, there is little change in overall prevalence after five or six allergens have been included in the panel. , although less is known about other countries, mite allergens appear to be widespread in tropical and subtropical areas. for the most part, skin tests and serologic tests for sensitization give similar results when technical failures and differences between allergens are taken into account. however, they are not equivalent. skin tests also depend on the ability of mast cells to degranulate and for the skin to respond to histamine. when skin test results are negative, clinical allergy is unlikely even in the presence of specific ige. modern attempts to define asthma start with the ciba guest symposium of on the terminology, definitions, and classification of chronic pulmonary emphysema and related conditions. the symposium defined asthma as "the condition of subjects with widespread narrowing of the bronchial airways, which changes its severity over time spontaneously or under treatment, and is not due to cardiovascular disease". it further identified the clinical characteristics as "abnormal breathlessness, which may be paroxysmal or persistent, wheezing, and in most cases, relief by bronchodilator drugs (including corticosteroids)." soon after the publication of this report, scadding, one of the contributors to the symposium, made two important points. first, what had been described as a definition in the report was not a true definition in that it did not provide a clear year definition ciba foundation condition of subjects with widespread narrowing of the bronchial airways, which changes its severity over short periods spontaneously or during treatment american thoracic society disease characterized by increased responsiveness of the trachea and bronchi to various stimuli and manifested by widespread narrowing of the airways that changes in severity spontaneously or as a result of therapy world health organization (who) chronic condition characterized by recurrent bronchospasm resulting from a tendency to develop reversible narrowing of the airway lumina in response to stimuli of a level or intensity not inducing such narrowing in most individuals american thoracic society clinical syndrome is characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli. major symptoms are paroxysms of dyspnea, wheezing, and cough, which may vary from mild and almost undetectable to severe and unremitting (i.e., status asthmaticus). primary physiologic manifestation of this hyperresponsiveness is variable airway obstruction, occurring in the form of fluctuations in the severity of obstruction after bronchodilator or corticosteroid use, or increased obstruction caused by drugs or other stimuli, as well as evidence of mucosal edema of bronchi, infiltration of bronchial mucosa or submucosa with inflammatory cells (especially eosinophils), shedding of epithelium, and obstruction of peripheral airways with mucus. nhlbi/nih lung disease with the following characteristics: ( ) airway obstruction that is reversible (but not completely in some patients) spontaneously or with treatment, ( ) airway inflammation, and ( ) increased airway responsiveness to a variety of stimuli. nhlbi/nih , chronic inflammatory disorder of the airways in which many cells play a role, particularly mast cells, eosinophils, and t lymphocytes. in susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and cough in early morning. symptoms are usually associated with widespread but variable airflow limitation that is at least partly reversible spontaneously or with treatment. inflammation also causes an increase in airway responsiveness that is associated with a variety of stimuli. nih/nhlbi chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. the chronic inflammation causes an increase in airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. these episodes are usually associated with widespread but variable airflow obstruction that is often reversible spontaneously or with treatment. nhlbi/nih, national heart, lung, and blood institute/national institutes of health. symptom questionnaires do not have the disadvantages of reported diagnoses, but they have problems of their own. first, used alone, symptoms are rarely diagnostic of a condition. this may not be a serious problem when there is no need for an accurate diagnosis in every case, but some symptoms are highly nonspecific. there may be considerable crossover of symptoms between different airway diseases such as asthma and bronchitis. second, the interpretation of similar symptoms may vary among different people. this may become a serious problem when making comparisons in the settings of different cultures and languages. in translating asthma questionnaires, there may be particular problems in translating terms such as wheeze when there may not be an equivalent word, and even people who speak the same language may interpret wheeze differently. given the lack of a gold standard to test these questionnaires against, their validity cannot be fully assessed, because it depends in part on whether they are seen as plausible indicators of the presence of asthma and standardization against a plausible alternative indicator. in the iuatld questionnaire, this indicator was the airway response to histamine, which usually increases tests. they have some of the same limitations as reversibility testing. more promising has been the use of bronchial challenge tests, most of which use a direct bronchoconstrictor such as histamine or methacholine. lung function is assessed before and after inhaling increasing doses of the agent. the decline in lung function (usually the forced expiratory volume in second [fev ]) is regarded as a marker of asthma. , this may be expressed as the dose or concentration of agent that produces a given (often %) fall in lung function, in which case the result usually is dichotomized as those falling by at least that amount (i.e., hyperresponsive) and those that do not (i.e., normal). alternatively, the slope of the dose-response curve has been used as a continuous measure of airway reactivity, a method that uses epidemiologic information more efficiently but may be clinically less intuitive. development of these tests for use in surveys has provided a tool for assessing a physiologic measure associated with asthma. there is little difference between the use of histamine and methacholine, but methacholine is more widely used because it has fewer side effects. one disadvantage of nonspecific challenge tests is that they produce positive results for those with asthma and also with chronic obstructive pulmonary disease (copd). , this has led to the use of alternative agents that act indirectly by releasing mediators from mast cells in the airway. challenge agents include adenosine, hypertonic solutions (e.g., saline, mannitol), exercise, and cold, dry air. these alternatives have not been used as widely as methacholine. exercise testing usually has been confined to studies of children. its effects depend on weather conditions (e.g., cold, dry conditions produce a greater stimulus than warm, moist conditions), and it requires well-motivated groups of participants. equipment to provide cold, dry air has not been widely available. use of saline and mannitol has promise, but they have not been widely used in surveys. the theoretical advantage of using these methods is that they are less likely to provoke airway constriction in those with copd. allergic rhinitis has been investigated much less frequently than asthma using epidemiologic approaches. population-based studies are made difficult by misclassification arising from reliance on questionnaires to establish the presence of allergic rhinitis. typically, the questionnaires used by epidemiologists ascertain self-reports of responders having something they call allergic rhinitis or hay fever. nonetheless, studies show that allergic rhinitis is among the most common chronic diseases. symptoms of individuals with rhinitis include sneezing, nasal irritation, rhinorrhea, and nasal blockage. these symptoms can also involve the eyes, ears, and throat, including postnasal drainage. allergic rhinitis is most commonly classified as seasonal, perennial, or occupational, but a recent guidelines statement advocated classifying allergic rhinitis as intermittent or persistent. the symptoms of allergic rhinitis are associated with exposure to allergen sources such as pollens, pets, and house-dust mites (hdms). symptoms result from inflammation induced by a specific ige-mediated immune response to the allergens. criteria for diagnosing chronic rhinosinusitis have been published. , a questionnaire based on the symptomatic part of this definition has been devised and tested for epidemiologic surveys. in patients with asthma. although it is not diagnostic of asthma, it is reassuring to find that answers to the questionnaires can predict the results of the alternative test and that they can do this in approximately the same way in different countries and different translations. for the isaac questionnaires, a video was developed that demonstrated the symptoms of asthma, and it was used to help standardize comprehension of the questionnaire in different settings. although fully validated questionnaires for diagnosing asthma are not available, the current questionnaires do allow comparison of symptoms that plausibly represent conditions close to asthma in a standardized way. although cautious interpretation is always advisable, they have enabled substantial advances in our knowledge of the relative distribution of the condition. an objective test for asthma that did not depend on interpretation of questionnaires would be ideal, and several tests have been proposed. the lack of a gold standard for diagnosing asthma and the similarity of asthma to other conditions make a perfectly validated test unattainable, but tests do provide additional tools to check the findings of surveys that use questionnaires only. the physiologic tests for asthma have been based on the definition of asthma as a condition of the airways that changes its severity over time spontaneously or after treatment (box - ). reversibility of airway obstruction after use of a bronchodilator (i.e., reversibility testing) has been used in clinical studies to distinguish between asthma and fixed airway obstruction, and some have used it as a test in surveys to identify asthma. the difficulty lies in interpreting the results. a positive test result indicates the likely presence of asthma, but a negative test result is uninformative. because a patient with asthma who is receiving good treatment or in remission for some other reason does not respond to a bronchodilator, this approach has not found much use in surveys of the general population. spontaneous changes in airway caliber can be assessed using peak flow diaries, a clinical technique that has been commonly used in primary care in the united kingdom. although they can be difficult to use in large-scale studies, they do provide data comparable to that using more invasive bronchial challenge kingdom, and the netherlands. low prevalence rates were found in spain, iceland, and italy. the second phase of the isaac study estimated the prevalence of positive skin-prick test responses to at least one of six allergens in children between the ages of and years living in sites, mostly in western europe. , estimated prevalence ranged from . % in tallin, estonia, and . % in mumbai, india, to . % in rome, italy, and . % in almeira, spain. unlike in western countries, the prevalence of sensitization in africa heavily depends on the methods used to assess sensitization. in rural areas, the prevalence of positive skin-prick test results is very low, whereas the prevalence of allergen-specific ige is high. the high prevalence of allergen-specific ige in poor rural areas was first shown in zimbabwe (formerly called southern rhodesia) by merrett and associates, but the dissociation in these environments between specific ige levels and skin test results also has been shown in kenya and south africa. where skin test results are negative, even in the presence of specific ige to aeroallergens, clinical allergy is rare. asthma is a global problem. it is estimated that approximately million people worldwide have asthma. prevalence rates for children and adults are substantially different in countries around the world. the first phase of the isaac study provides the most extensive information on variation in childhood asthma prevalence. in , the isaac steering committee reported findings for , -to -year-old children ( centers in countries) and , -to -yearold children ( centers in countries). for the younger and older children, the prevalence of asthma symptoms was based on a positive response to this question: have you had wheezing or whistling in the chest in the past months? across countries, there was an approximately twentyfold range of prevalence, with the highest rates usually found in more developed countries (figs. - and - ) . the countries with the highest prevalence rates (> %) were the isle of man, the united kingdom, new zealand, ireland, australia, peru, panama, costa rica, the united states, and brazil. the ecrhs assessed geographic variation in asthma among , adults from countries. a sixfold variation in the prevalence of current asthma was found among the countries. a high (> %) prevalence of asthma was found in australia, new zealand, the united states, ireland, and the united kingdom. asthma prevalence of less than % was found in iceland, parts of spain, germany, italy, algeria, and india. current asthma was defined in the ecrhs as "having an attack of asthma in the past months or currently taking medicine for asthma." the ecrhs did not examine many sites outside the developed market economies, but the world health survey interviewed adults older than years of age in six continents using questions derived from the ecrhs on wheezing and diagnosed asthma. the prevalence of diagnosed asthma ranged from . % in vietnam to . % in australia (fig. - ) . a very wide variation in the prevalence of diagnosed asthma (and wheezing) was found in all countries, regardless of gross national income per capita adjusted for purchasing power parity. in countries eczema similar to allergic rhinitis, the epidemiology of eczema is less well understood than the epidemiology of asthma. eczema, also known as atopic dermatitis, is a pruritic rash characterized by chronic, recurrent papular lesions typically affecting skin at the flexor surfaces, buttocks, and back of the neck. infants frequently have involvement of the face. in its acute and subacute forms, eczema is characterized primarily by erythema and a papular eruption, but in its chronic form, it is characterized by lichenification of affected areas. allergic sensitization plays an important role in provoking eczema flares, particularly in pediatric patients. some studies have relied on physician diagnosis to define eczema, but standardized questions have been developed for identifying eczema cases with or without additional information from standardized examination. these questions are included in the isaac questionnaire, and they focus on the chronic and recurrent nature of the rash, its location, and the presence of pruritus. during the past years, food allergy has received increased attention, and there is a growing body of literature available regarding its epidemiology. food allergy is an immune-mediated reaction to a food. it can produce a wide spectrum of clinical manifestations, including acute ige-mediated reactions, mixed ige-mediated and non-ige-mediated reactions that are often characterized by insidious gastrointestinal symptoms, and non-ige-mediated syndromes such as allergic colitis and food protein-induced enterocolitis syndrome. even among patients with acute ige-mediated types of food-allergic reactions, symptoms can vary and include one or many of the following: urticaria, angioedema, pruritus, cough, wheezing, hoarseness, vomiting, diarrhea, oral pruritus, hypotension, and rhinorrhea. because the diagnosis is based on the clinical history and diagnostic test results, with the gold standard being a double-blind, placebo-controlled food challenge, conducting large epidemiologic surveys can be difficult because of reliance on questionnairebased tools for identification of food allergy and evidence of ige sensitization. because there is no validated questionnaire for food allergy and many reported food allergies are not confirmed when a full diagnostic evaluation is completed, estimates obtained from questionnaires are likely to be inflated. the prevalence of sensitization depends on the selection of allergens. for this reason, the relative prevalence of responses to a standardized panel of allergens is more informative than an absolute prevalence. the ecrhs estimated the prevalence of specific ige (≥ . ku/l) to mites (dermatophagoides pteronyssinus), cats, grass (timothy grass), or cladosporium among young adults between the ages of and years in centers, mostly in western europe. the prevalence of a positive response to any of the four common allergens ranged from . % to . % ,with a median prevalence of . %. high prevalence rates were found in australia, new zealand, the united states, the united rates for different questionnaire-based indicators of asthma: physician report, current disease, and the symptom of wheezing used in the national health and nutrition survey of the united states from through . questions that ask about asthma or wheeze provide estimates that are almost twice those of questions asking about either alone, and this difference varies with age. for those between the ages of and years, with the lowest incomes (<us$ ), none of sites had a prevalence of diagnosed asthma of more than %; among countries with intermediate incomes, ( %) of countries had a prevalence higher than %; and among counties with the highest incomes (<us$ ), ( %) of countries had a prevalence above %. the national health interview survey (nhis) and the national health and nutrition examination survey (nhanes) include random samples of the whole u.s. population. both also provide periodic prevalence estimates. standardization of the methods of prevalence surveys is essential because the wording of questions on asthma or other aspects of data collection may affect estimates based on the questionnaires. for example, table - provides prevalence country wheeze is much more likely to be associated with a diagnosis of asthma. state-specific asthma prevalence data for adults are available from the behavioral risk factor surveillance system (brfss), a state-based, random-digit-dialed survey of noninstitutionalized u.s. adults age years or older. it was conducted by the centers for disease control and prevention (cdc) and the state or territorial departments of health. information was gathered from all states, puerto rico, the virgin islands, and the district of columbia. the definition for current asthma was based on providing positive answers to two questions: have you ever been told by a doctor, nurse, or other health professional that you had asthma? do you still have asthma? the overall current asthma prevalence rate was . %, ranging on the mainland from % in tennesee to . % in vermont ( fig. - ) . asthma prevalence rates in the united states vary with demographic and social conditions. figure - gives prevalence rates from the nhis for through . children have more reported asthma than adults, women have more than men, those in poverty have more asthma than those not in poverty, asian respondents reported less asthma than any other group, and all other racial groups have more asthma than white respondents. estimating incidence and prevalence of allergic rhinitis is as challenging as for asthma. the diagnosis is symptom based, and there is no consensus for measurement. some attempts have been made to estimate the incidence of allergic rhinitis using cohort or longitudinal data. in a birth cohort study from germany that followed children for years, % of them developed seasonal allergies, defined as a combination of exposure-related symptoms and sensitization. a % cumulative incidence rate was determined in a -year longitudinal study of college students. the researchers also observed that regression of hay fever symptoms was inversely related to age of onset. almost % of those with hay fever onset before years of age had improvement of their symptoms. the isaac researchers collected data on -month prevalence of allergic rhinoconjunctivitis for children to years old from countries. allergic rhinoconjucntivitis was defined as having a problem with sneezing or a runny or a blocked nose without a cold or flu and accompanied by itchy, watery eyes. there was a thirtyfold variation in the prevalence rate among the sites, from . % to . %. estimates for adults obtained in the ecrhs ranged from . % in algeria to . % in australia. the median prevalence of nasal allergies in the ecrhs was about %. countries with significantly high prevalence rates were the netherlands, belgium, switzerland, france, the united kingdom, new zealand, australia, and the united states. chronic rhinosinusitis is an inflammation of the nose and paranasal sinuses lasting over weeks. another multicenter european study estimated the prevalence of chronic rhinosinusitis using a questionnaire based on the european position paper on rhinosinusitis and nasal polyps (ep os) on symptom criteria to be about % among adults, ranging from % in helsinki, finland, to % in coimbra, portugal. the condition was more common in women, in younger participants, and in smokers. the worldwide prevalence of eczema was best documented in isaac phase three, in which the prevalence for -to -year-old children ranged from . % in india to . % in ecuador. , the worldwide prevalence of eczema in children to years of age has increased in most regions of the world and has remained stable only in western, northern, and eastern europe. for this younger age group, the prevalence in isaac phase three was . %, an increase from the prevalence in phase one of . %. the overall prevalence among -to -year-old adolescents declined to . % in isaac phase three from . % in phase one. the regions with the most striking increases in eczema prevalence were asia and latin america. a growing number of population-based studies have estimated food allergy prevalence and incidence. the prevalence of food allergy and the foods that are most commonly implicated in food allergy vary widely across the world. for example, a metaanalysis found that the self-reported prevalence of food allergy ranged from % to %. although some of the heterogeneity observed is attributable to study methodology, other studies that have focused on specific geographic regions and populations support the notion that there is marked worldwide heterogeneity in food allergy prevalence. for example, in the united states, the estimated prevalence of food allergy is % to % among children, , and in australia, a prevalence of more than % among -year-old children has been reported. in contrast, findings from a population-based birth cohort from the united kingdom indicate that between . % and . % of infants have food allergy in the first year of life, and in other u.k. studies, the estimated prevalence of food allergy among -year-old children is . % to . %, and among adolescents, it is . %. an international study that used allergen-specific ige data for young adults confirmed the heterogeneity of food allergy prevalence observed in the questionnaire-based studies. in this study, prevalence of sensitization to any of foods ranged from . % in iceland to . % in the united states. however, sensitization to the major foods, such as fish, egg, and milk, was uncommon (< %). the incidence and prevalence of allergy to specific foods have been studied. a random telephone survey administered in to estimate the prevalence of seafood allergy in the united states found that . % of the general population reported allergy to fish or shellfish. another telephone survey was conducted in and repeated in and to estimate the prevalence and incidence of peanut and tree nut allergies. , the prevalence rate of peanut or tree nut allergy was stable at . % among adults, but it increased from . % to . % to . % among children during this period. in contrast, data from u.k. general practices were used to estimate a prevalence rate of peanut allergy of . %, and a school-based survey in singapore and the philippines estimated a prevalence rate of peanut and tree nut allergy of less than %. in australia, the prevalence of peanut allergy may be higher than that observed in the united states or united kingdom, because the prevalence of peanut allergy among -year-old children was estimated to be %. there is limited information about the epidemiology of allergic gastrointestinal diseases. however, the incidence and prevalence of eosinophilic esophagitis appear to have increased over the past decade. a range of gastrointestinal symptoms and mucosal eosinophilia characterizes eosinophilic esophagitis. approximately one half of cases demonstrate ige sensitivity to foods. what was once thought to be a rare disease was found people born later in the century having a higher prevalence of disease throughout their lives. in cross-sectional surveys, this pattern of disease is indistinguishable from a decline in disease with age. in the ecrhs, there was almost no net change in the prevalence of sensitization over the years between the surveys within any birth cohort, but succeeding birth cohorts had increasingly high prevalence rates. a subsequent analysis of some british data showed that this phenomenon was apparent in successive birth cohorts since at least the s. to evaluate changes in prevalence, repeated surveys are required for the same populations and using the same instruments for assessment. in the s and s, smith in birmingham, england, first observed that the prevalence of asthma was rising among schoolchildren undergoing school medical examinations. since then, many studies have assessed changes in prevalence. these studies are summarized in figure - , which by one study based in switzerland to have increased in incidence from . to . per , adults from to . , in another study in hamilton county, ohio, the incidence in children younger than years old has increased from . to . % per year, and the prevalence has increased from . % to . % from to . a few studies have made serial assessments of the levels of ige specific to common allergens in the same populations over many years. these studies have shown a fairly consistent increase in the prevalence of sensitization over time. analysis of the data from the tucson and the first and second ecrhs surveys found that the apparent decline in the prevalence of sensitization with age was more easily explained as a birth cohort effect. the apparent effect of age results from . year of survey germany in two surveys made very close together in time and seem unlikely to represent a long-term trend. the isaac reassessed prevalence of asthma and other allergic diseases by questionnaire for -to -year-old children in centers in countries and for -to -year-old children in centers in countries. the results for asthma prevalence are given in figure - . among the younger children, rates were more commonly rising than declining, whereas among the older children, the rising and declining rates were more evenly balanced. among the older children in centers with a high prevalence, the rates were more likely to be declining. in the united states, the nhis, a population-based interview survey of u.s. households, is a key source of information on trends in the prevalence of asthma. before , prevalence estimates were based on a positive response to one question: during the past months, did you have asthma? in , the nchs redesigned the nhis questionnaire. consequently, answers to the nhis questionnaire after cannot be compared with those in previous surveys because the new questions assess asthma morbidity differently. lifetime incidence is measured by this question: have you ever been told by a doctor or other health professional that you had asthma? the prevalence of asthma attacks is obtained by a positive response to another question: during the past months, have you had an episode of asthma or asthma attack? the asthma attack prevalence in was . % for all age groups. in , nchs added a question (do you still have asthma?) after the lifetime incidence question to provide estimates of current asthma prevalence (table - ). the estimates indicate that . % of u.s. residents currently have asthma that has been diagnosed by a doctor or other health professional. table - provides the estimates for asthma prevalence before and after the revisions of the nhis questionnaire. figures - and - show data from the nhis with different estimates of prevalence of asthma over time for children and adults and by race or ethnic group. for children and adults, the -month and lifetime prevalence rates increased over time. current and attack prevalence rates have not shown a marked trend since the use of these outcomes began. prevalence rates also vary among racial and ethnic groups; this variation likely reflects differences in genetic, environmental, social, shows the changes in prevalence over time plotted on a logarithmic scale. most of the studies surveyed children, although a few looked at military conscripts in countries with compulsory military service. the very large variations in the absolute prevalence estimates reflect differences in the definitions used and the populations examined. from the late s to the mid- s, the values rose uniformly at a rate that represented a doubling of prevalence approximately every years, and this occurred regardless of the definition used and the starting prevalence. only in the mid- s did reports begin to suggest that this increase had slowed down. a much smaller number of studies have assessed changes in the prevalence of airway responsiveness ( fig. - ). during the s, a small number of studies showed increases in prevalence over time. [ ] [ ] [ ] [ ] the study of dubois, which showed a yearly increase in prevalence among belgian conscripts over years, is particularly persuasive given the difficulties of standardizing the tests. since the s, most studies have shown a flattening of or a decline in prevalence, [ ] [ ] [ ] although increases were seen in a study from ghana, and a large increase was observed in year prevalence (%) asthma period prevalence current asthma prevalence asthma attack prevalence and cultural influences among population groups. between and , the prevalence of asthma had increased from . % to . % among whites and from . % to . % among blacks, representing cumulative increases of approximately % per annum. between and , rates of current asthma had increased from . % to . % among whites, a cumulative increase of approximately . % per annum, and from . % to . % among blacks, a further increase of approximately % per annum (see fig. - ). many of the studies that reported changes in the prevalence of asthma and wheeze also found increased reporting of other allergic diseases, most notably allergic rhinitis and eczema in children. as for asthma, almost all studies reporting before the mid- s found increases in other atopic conditions with time. since then, results have been more mixed, and the isaac has provided a more representative spread of data than is possible from individual studies. (fig. - ) for the -to -yearold children, most sites showed an increase in prevalence of allergic rhinoconjunctivitis and eczema in the years after the baseline surveys of isaac phase one. for the -to -year-old children, the changes were more evenly distributed between increases and decreases, and the size of the annual percentage change (positive and negative) appeared to be proportional to the initial value. a follow-up of the ecrhs sample approximately years later estimated an incidence rate of rhinitis of . cases per thousand people per year. males had a greater risk in childhood and a lower risk in adulthood of incident rhinitis compared with females. atopic subjects had the highest incidence of rhinitis, at more than cases per thousand people per year. were compared for fathers ( . %) and sons ( . %) and for mothers ( . %) and daughters ( %). later findings from a u.k. study suggested that the prevalence of hay fever had been stable in the latter half of the s. the worldwide prevalence of eczema among -to -year-old children has increased in most regions of the world and has remained stable only in western, northern, and eastern europe. for this younger age group, the prevalence found in the isaac phase three was . %, an increase from the prevalence in phase one of . %. the overall prevalence among -to -year-old children declined to . % in the isaac phase three from . % in the isaac phase one. the regions with the most striking increases in eczema prevalence were asia and latin america. two cross-sectional chinese studies from chongqing showed a statistically significant increase in food allergy prevalence, from . % in to . % in , among young children between the ages of and months. these studies also showed that sensitization to food allergens assessed by positive skin-prick test results had almost doubled, rising from . % to % during the same -year period. other than these studies, there is little evidence on time trends using food challenge tests in general populations, although there is more circumstantial evidence for an increase over time. the prevalence of allergic rhinitis has increased over time in the united states. the nhis estimated a seasonal allergic rhinitis prevalence of %, , an increase of % from the prevalence estimate of . % based on the nhis. the nhis estimated that about . million americans, . million of them younger than age years, have allergic rhinitis. , the survey also showed geographic variation in the prevalence of seasonal allergic rhinitis, with the highest estimated prevalence for the western united states ( . %), followed by the northeast ( . %), south ( . %), and midwest ( . %). studies in other countries have also documented an increase in the prevalence of allergic rhinitis over time. von mutius and colleagues observed a significant increase in hay fever prevalence among schoolchildren from leipzig, germany. the hay fever prevalence rate in the - survey was . %; in the - survey, the prevalence had increased to . %. an italian retrospective study compared data on allergic rhinitis prevalence among italian recruits observed in and from to . the investigators found a significant increase in allergic rhinitis prevalence, from . % in to . % in the - period. results from surveys conducted in and of an adult danish population showed a significant increase of % in the prevalence of allergic rhinitis after adjustment by age and gender. a scottish study observed a threefold increase in the age-adjusted prevalence rate of hay fever between and and in middle-age adults. the hay fever prevalence rates change per year (%) mean prevalence (%) eczema d some genotypes were specifically associated with childhoodonset asthma. gender data from epidemiologic studies consistently reveal male gender to be a risk factor for asthma among young children, whereas studies of adolescents and adults show females to be at greater risk. a review of prospective studies showed that male children were at greater risk for incident asthma or wheeze, and studies of adolescents or adults showed females to be at greater risk. , one hypothesis explaining the excess of asthma seen in boys during childhood is the different airway geometry in boys and girls. boys have smaller airways for a given lung size than girls. lower flow rates were found for boys to years of age, and they have higher airway resistance compared with girls of the same age. this difference in airway anatomy predisposes boys to more wheezing, and they have a higher incidence of lower respiratory tract infections. , martinez and coworkers suggested that reduced lung function was a key determinant of wheezing in boys and girls. in the tucson study, lung function was measured within weeks of birth in healthy infants. the subsequent incidence of wheezing illness over the first years of life was increased in boys with high respiratory resistance and in girls with low functional residual capacity. one hypothesis for females being at higher risk of developing asthma or wheeze during adolescence or adulthood is the smaller relative airway caliber in women compared with men after puberty. epidemiologic studies consistently show an association between atopy and asthma, although the strength of the association varies with the index used and the study population. part of the problem lies in the ambivalent nature of the term asthma.the symptoms of asthma in older adults also can indicate copd, and the differential diagnosis may be difficult even in a clinic. a distinction used to be made between asthma and other wheezy syndromes in children. however, the management is very similar, the distinction has fallen largely into disuse, and use of the term nonatopic asthma has become common for children, whereas it was prviously reserved for a condition associated with eosinophilic inflammation but negative skin test results that predominantly presented in middle age, often among women, and had a relatively poor prognosis. the idea that atopic and airway components of asthma are distinct , has been confirmed by genetic studies thatidentified gene polymorphisms associated with asthma but not obviously related to the regulation of ige or associated immunologic responses. isaac found little variation in the prevalence of nonatopic asthma that could be explained by the local gross income, but prevalence of asthma associated with sensitization became less common in low-income settings. , the distinction is important, because atopic asthma in children apears to have a less benign course than nonatopic disease. burrows and colleagues assessed this association in a random sample selected from tucson, arizona, using skin tests with local antigens as an index of atopic status. in children to years old, atopy was strongly associated with attacks of wheezing and dyspnea, regardless of whether asthma had been diagnosed. later analyses of the tucson population indicated that the total serum ige level was more strongly associated with the prevalence of asthma, an association that was also found in an nhanes analysis (discussed later). another study using self-reported during the -year period from to , hospital admissions for food allergy showed continuing increases of to cases per million people overall and to cases per million children, particularly in the -year-old age group. hospital admissions attributed to "dermatitis due to food" increased in england and wales from cases per million people in to cases per million people in , an increase of almost % per annum. , apart from true changes in the epidemiology of food allergy, alternative explanations for this dramatic increase in health care use for presumed food allergy include changes in the coding behavior of doctors, changes in the awareness and behavior of patients, and changes in the health care system. in the united states, the nhis recorded much smaller increases in parent-reported food allergy, although such reports are usually regarded as unreliable. increasing reports of more specific complaints of peanut allergy have been made for children in the united states and the united kingdom. a genetic component of asthma has long been recognized, based initially on the observation that asthma tends to cluster in families. familial aggregation, twin, genetic linkage, and association studies subsequently provided evidence to support this hypothesis. detailed descriptions of the molecular genetics of asthma and atopic disease are provided in chapter . family aggregation studies compare disease occurrences between family members. disease aggregation between siblings and between parents and children supports a hereditary component, whereas increased aggregation between spouses suggests that environment is more important. however, aggregation among related individuals may represent shared genes or a common household environment. evidence of a hereditary component of asthma has been demonstrated in numerous family-based studies. [ ] [ ] [ ] [ ] population-based studies also have shown an increased prevalence of asthma among first-degree relatives of index cases. [ ] [ ] [ ] [ ] [ ] twin studies compared disease frequency in monozygotic (mz) twins, who share % of their genes, with dizygotic (dz) twins, who share on average % of their genes. this is another method to determine the relative contribution of genetic and environmental factors. a greater occurrence of disease in mz than in dz twins is evidence for a genetic component to the disease because both types of twins are assumed to share environmental factors similarly. significantly higher concordance rates for mz twins have been observed in some small studies. larger studies of twin registries also suggest a significant genetic contribution to asthma. in twin pairs from the swedish twin registry, edfors-lubs found the prevalence of asthma to be . %. the higher concordance of asthma in mz ( . %) over dz ( . %) pairs suggests that genetic factors contribute to asthma. similarly, duffy and associates observed a % concordance in mz twins and % in dz twins in a total of australian pairs. whole-genome association studies have identified specific loci associated with clinical asthma. the genes associated with total ige levels were not strongly associated with asthma, and many related methodologic issues complicate the study of diet and disease. obtaining unbiased exposure estimates is difficult because of recall problems, difficulties in measuring intake, and limited range of variation in the western diet. diet is fundamental to existence and is closely associated with many lifestyle, social, cultural, and environmental factors that may influence the development of disease. although many studies adjust for potential confounders, it has been argued that this is insufficient and that many or all of the reported associations between diet and disease are likely to be confounded by social and behavioral factors acting across the life course. , several reviews on the topic of diet and asthma and allergic disease are available. [ ] [ ] [ ] [ ] [ ] [ ] [ ] observational and randomized studies have directly correlated dietary sodium levels with increased bronchial responsiveness to histamine challenge. [ ] [ ] [ ] one study showed this response occurred in males but not in females. ecologic data from england and wales support these experimental findings; the purchases of table salt correlated with the regions of increased asthma mortality for men and children. other population-based studies, however, have not shown a relationship between urinary sodium levels and methacholine airway responsiveness, [ ] [ ] [ ] although two did show an association with higher levels of potassium excretion. , britton and associates found that increased magnesium intake was associated with a reduced risk of bronchial hyperresponsiveness and wheeze. because of the relationships among sodium, potassium, and magnesium, these investigators suggest the possibility that low levels of dietary magnesium may confound the association of sodium and potassium with bronchial hyperresponsiveness or asthma. a systematic review of the clinical trials of dietary sodium reduction in persons with asthma concluded that dietary sodium reduction does not significantly improve asthma control. dietary sodium reduction was associated with some improvement in lung function in people with exercise-induced asthma, but because extremely large reductions in sodium intake were required, the clinical utility, as distinct from the potential public health relevance of this finding, is likely to be small. a systematic review of the studies correlating asthma and allergic disease in children with dietary intake or status of vitamin a, vitamin c, vitamin e, selenium, zinc, copper, iron, fruits, vegetables, and components of the mediterranean diet identified eligible reports. there were no randomized, controlled trials, and all studies were judged to be at moderate to substantial risk of bias. meta-analyses demonstrated that serum vitamin a was lower in children with asthma compared with controls (odds ratio [or] = . ; % confidence interval [ci], . to . ). the meta-analyses also showed that high maternal dietary vitamin e intake during pregnancy was protective against development of childhood wheezing (or = . ; % ci, . to . ) and that maternal adherence to a mediterranean diet during pregnancy was protective against persistent wheeze (or = . ; % ci, . to . ) and atopy (or = . ; % ci, . to . ) in children. seventeen of fruit and vegetable studies reported beneficial associations with asthma and allergic outcomes. although weak, the available epidemiologic evidence supports dietary intake of vitamin a, vitamin d, vitamin e, zinc, fruits, vegetables, and a mediterranean diet for the prevention of asthma. the fatty acid composition of the diet, particularly the relative amounts of n- (ω- ) and n- (ω- ) polyunsaturated fatty diagnosis of hay fever in a population sample from western pennsylvania showed atopy to be more common in asthmatic than nonasthmatic children. two other studies demonstrated parental atopy and asthma as risk factors for asthma in their children. , the results also demonstrated a complex interaction between these factors and the gender of the child. a study of new zealand children assessed the relationship of total serum ige levels at the age years and asthma, and investigators found that the prevalence of diagnosed asthma was strongly related to the serum ige level (p trend < . ). , asthma was not reported for children with ige levels of less than iu/ ml, whereas % of children with ige levels of iu/ml or higher had asthma. asthma and atopy are closely linked, and the causal nature of the factors seems to be complex and require more investigation. nonetheless, atopy in parents and children does increase the risk of asthma and can be used as an empiric predictor. diet diet, including breastfeeding, food intolerance and sensitivity, and intake levels of specific micronutrients, has been investigated as a potential risk factor for asthma for over decades. in , burney was among the first to propose diet as a potential explanation for the rising frequency of asthma, suggesting the possibility that increasing salt intake might have a role. the rationale for investigating diet as a possible risk factor for asthma and allergic disease is that diet has changed in recent decades and biologically plausible mechanisms exist. many dietary constituents have been investigated for potential involvement in the pathogenesis of asthma and allergic disease, including antioxidants (e.g., vitamin e, vitamin c, carotenoids, flavonoids), polyunsaturated fatty acids, vitamin d, folate, , foods (e.g., fruits, vegetables), metals or trace minerals (e.g., sodium, magnesium, selenium, zinc, copper), and components of the mediterranean diet ( increased ige levels, eosinophilia, methacholine responsiveness, asthma-related hospitalization, exacerbations, use of antiinflammatory medication, and use of oral corticosteroids and with reduced asthma control scores. analogous findings have been reported for adults with asthma. similarly, for children with atopic dermatitis, lower serum -oh-d concentrations have been associated with increased severity of atopic dermatitis. longitudinal cohort studies quantifying vitamin d status before the development of disease are not subject to many of the limitations of cross-sectional studies, such as reverse causation, whereby the vitamin d status of an individual is influenced by the presence of disease (e.g., children with asthma may spend more time indoors). a systematic review and metaanalysis of the longitudinal studies relating maternal vitamin d status during pregnancy to childhood outcomes concluded that high maternal dietary vitamin d intake is associated with a reduced risk of children wheezing up to the age of years (or = . ; % ci, . to . ). however, maternal dietary vitamin d intake during pregnancy is not indicative of total body vitamin d status, because % of vitamin d is derived from cutaneous exposure to ultraviolet light. in contrast to maternal dietary vitamin d intake, high (> nmol/l) maternal serum -oh-d levels in late pregnancy have been associated with an increased likelihood of childhood eczema at age months and asthma at age years. during infancy, increased vitamin d intake has been associated with an increased risk of atopic dermatitis at age years and an increased likelihood of allergic rhinitis and atopic sensitization at the age of years. , in later childhood, an increased serum -oh-d concentration at years of age has been associated with a reduced likelihood of asthma at years of age, and an increased serum -oh-d concentration at years of age has been associated with a reduced likelihood of asthma, rhinoconjunctivitis, and atopic sensitization at years of age. the epidemiologic data support the hypotheses that vitamin d may have beneficial and adverse influences on the development of asthma and allergic disease. ongoing clinical trials are clarifying the potential clinical role of vitamin d in modifying the risk of developing asthma and as an adjunct to asthma and atopic dermatitis therapy. although breastfeeding of infants is recommended because of well-documented benefits for mother and child, the effects of breastfeeding on the subsequent development of atopic dermatitis, wheezing disease, and asthma are not clear. , conceptually, the advantageous consequences of breastfeeding for the infant include acquisition of maternal antibodies and immune-competent cells such as macrophages and leukocytes and protection against early occurrence of lower respiratory tract infections. however, breastfeeding may also be a route of exposure to a variety of immunologically active substances from the mother, such as tobacco smoke, cow's milk, eggs, wheat, maternal ige, and sensitized lymphocytes. many studies have investigated the association between breastfeeding, asthma, wheezing illness, and atopic disease, and they have been subject to several systematic reviews, most of which highlight the limitations and difficulties in conducting and interpreting such studies (e.g., confounding, recruitment bias, reporting bias, reverse causation, variation in breastfeeding patterns, inability to randomize and blind). the systematic reviews have themselves been reviewed in consensus documents, which conclude that the exclusive breastfeeding for to months of acids (pufas) found in fish and vegetable oils, respectively, affects cell functioning. fatty acids appear to have specific roles in inflammatory and immune responses, and changes in fatty acid consumption are a postulated cause of the rising incidence of asthma and other allergic diseases. , conflicting observational data relating n- and n- pufa intake or status during pregnancy, childhood, and adulthood to asthma and allergic disease have been surpassed by intervention trials. a systematic review with meta-analysis evaluated the interventional studies of n- and n- pufa supplementation in the context of primary prevention of asthma and allergic disease. ten reports from six double-blind, randomized, controlled trials were identified. four studies compared n- pufa supplements with placebo, and two studies compared n- pufa supplements with placebo. the meta-analyses failed to identify any consistent or clear benefits associated with n- pufa supplementation during pregnancy or infancy for atopic dermatitis two subsequent trials reported the consequences of n- pufa supplementation during pregnancy. in the first, highdose n- pufa supplementation of pregnant women from weeks' gestation and during breastfeeding reduced the incidence of food allergy and ige-associated atopic dermatitis in children in the first year of life compared with placebo ( % versus % [p < . ] and % versus % [p < . ], respectively). in the second, larger study of pregnant women, high-dose n- pufa supplementation from weeks' gestation until delivery did not reduce the incidence of ige-associated disease or atopic dermatitis during the first year of life compared with placebo (rr = . [ % ci, . to . ] and rr = . [ % ci, . to . ], respectively). there is insufficient evidence to recommend pufa supplementation in any period of life as a means of reducing the burden of asthma and allergic disease. the role of vitamin d in the cause asthma and allergic disease remains unclear. the increase in asthma and allergic disease in developed countries has been attributed to early-life vitamin d supplementation as rickets prophylaxis, and widespread vitamin d deficiency is thought to be a consequence of more time being spent indoors and the active promotion of sun avoidance. cross-sectional, observational studies have reported vitamin d status to be no different or increased in adults with asthma but decreased in children with asthma. , blood levels of -hydroxyvitamin d ( -oh-d) concentrations were found to be lower in adults with atopic dermatitis and allergic rhinitis. , in two studies using nhanes data, blood levels of -oh-d have been no different in adults with evidence of atopic sensitization; however, atopic sensitization was associated with reduced blood -oh-d levels in children and adolescents in one study but not in adolescents in the other. the effect of blood -oh-d levels on current wheeze depended on age and atopic status in another study using nhanes data, with nonatopic individuals and adults years of age or older having a greater risk of wheeze if they had lower -oh-d levels. in children with asthma, lower blood levels of -oh-d have been associated with increased asthma severity, including have reported associations between the prevalence of asthma and obesity, it is not possible to exclude reverse causation, whereby asthma may contribute to obesity through inactivity and use of oral corticosteroids. the most relevant data come from prospective cohort studies that have assessed risk for incident asthma in relation to initial weight or bmi. beuther and sutherland systematically reviewed prospective studies evaluating the association between bmi and incident asthma among adults. meta-analysis of the data from , subjects participating in the seven identified studies demonstrated that being overweight or obese (bmi ≥ ) was associated with an increase in the rate of -year incident asthma (or = . ; % ci, . to . ), with evidence of a dose effect for being overweight (or = . ; % ci, . to . ) or obese (or = . ; % ci, . to . ). there was no difference between sexes. a systematic review of similar literature for children and adolescents concluded that obesity precedes and is associated with the persistence and intensity of asthma symptoms. in observational designs, a potential methodologic concern is that nonspecific respiratory symptoms resulting from cardiorespiratory loading and deconditioning may be misclassified as asthma. careful studies of children and adults suggest that asthma is not inappropriately overdiagnosed in the obese. , observational studies have also reported adverse associations for bmi, obesity and overweight, and atopic dermatitis and atopic sensitization in children and adults. [ ] [ ] [ ] [ ] a retrospective case-control study of children with a mean age of . years confirmed an association between obesity and atopic dermatitis and reported that early-life and prolonged obesity was associated with atopic dermatitis. atopic dermatitis was more prevalent among children who were obese before years of age (or = . ; % ci, . to ) and between and years of age (or = . ; % ci, . to . ). obesity after the age of years was not associated with atopic dermatitis. children who were obese for . to . years (or = . ; % ci, . to . ) and for more than years (or = . ; % ci, . to . ) were more likely to be diagnosed with atopic dermatitis. infants at high risk for atopic disease reduces the likelihood of atopic dermatitis and that breastfeeding beyond to months appears to confer no additional benefit. , the available evidence also suggests that the breastfeeding of infants at low risk for atopic disease does not reduce the incidence of atopic dermatitis. the evidence for a protective effect of breastfeeding against respiratory disease is controversial. although breastfeeding appears to reduce the incidence of virus-associated wheezing episodes in young children (< years), the evidence of an effect on breastfeeding on the development of asthma is inconsistent. systematic reviews suggest that exclusive breastfeeding for to months is associated with a reduced risk of asthma in children to years old, but this beneficial effect is limited to infants at high risk for atopic disease. some systematic reviews have revisited the literature relating breastfeeding to childhood atopic dermatitis, asthma, and wheezing. a systematic review examining the association between exclusive breastfeeding for months or longer and the development of childhood atopic dermatitis identified reports from study populations and concluded that there was no strong evidence that exclusive breastfeeding confers a beneficial effect on the development of childhood atopic dermatitis (summary or = . ; % ci, . to . ), even in children at high familial risk (or = . ; % ci, . to . ). another systematic review clarified the association between breastfeeding and childhood asthma and wheezing after years of age. it examined publications and concluded that breastfeeding for months or longer did not confer any beneficial effect on the incidence of asthma and wheezing illness after the age of years. the summary odds ratio for any breastfeeding and wheezing was . ( % ci, . to . ), and for exclusive breastfeeding and wheezing, it was . ( % ci, . to . ). the prevalence of obesity increased dramatically in many countries, particularly western and other developed countries in the latter decades of the twentieth century. in the united states, for example, the prevalence of overweight and obesity among adults rose sharply across the s, such that most adults are now overweight. the prevalence of childhood overweight is also rising rapidly. the rise in obesity parallels the rise of asthma, and a hypothesis has been advanced that obesity could be a risk factor for asthma. several mechanisms have been postulated for the association, including the mechanical effects of obesity, a higher frequency of gastric esophageal reflux, upregulation of immunologic and inflammatory correlates of obesity, and a shared genetic basis for both conditions. , the association of obesity with asthma has been investigated in children and adults. camargo and colleagues offered one of the first reports in their paper based on the nurses' health study ii. the body mass index (bmi) in was positively and strongly associated with asthma risk over the next few years (fig. - ) . similar studies have addressed obesity and asthma in children. in a cross-sectional study using nhanes iii data, von mutius and colleagues found a positive association between bmi and asthma risk (or = . ; % ci, . to . ) by comparing the highest and lowest quartiles of bmi. in the tucson study, girls becoming overweight or obese between the ages of and years had a sevenfold increased risk for asthma. although many cross-sectional observational studies ≥ . at years. because most children are not hospitalized for lower respiratory tract disease, these results apply only to the more severe infections. a population-based study of children in east boston, massachusetts, however, found that a history of bronchiolitis or croup was a predictor of increased airway responsiveness. in another boston area study, children from a birth cohort with lower respiratory tract infection (i.e., croup, bronchitis, bronchiolitis, or pneumonia) in the first year of life were twice as likely to report two or more episodes of wheeze than children with no lower respiratory tract infection. the tucson children's respiratory study provides relevant data on follow-up from birth to age years. , results from this longitudinal study show that rsv infection was associated with an increased risk of infrequent and frequent wheeze by age years. the relative risk for wheeze after years of age for children with rsv infection compared with children with no rsv infection decreased over time. the relative risk decreased with age from . and . at age years to no risk at age years for infrequent wheeze and frequent wheeze, respectively. support for the idea that severe rsv-associated respiratory disease probably does not contribute to the development of asthma has been provided by a large ( pairs) danish twin registry study that applied genetic variance and direction of causation models to data on rsv-associated hospitalization and the development of asthma. a model in which asthma caused rsv-related hospitalization fit the data significantly better than a model in which rsv-related hospitalization caused asthma, suggesting that rsv infection does not cause asthma but reflects an underlying predisposition to asthma. the role of viruses in the natural history of asthma has been highlighted by several longitudinal cohort studies. the wisconsin childhood origins of asthma (coast) study prospectively evaluated the timing, frequency, severity, and cause of symptomatic viral infection in the first years of life in relation to later wheezing illness in a cohort of neonates at high familial risk for asthma. by using molecular technologies to identify viral infections in nasal lavage samples collected routinely and when symptomatic, this study highlighted the prognostic importance of hrv. having one or more hrvassociated wheezing episodes during the first years of life was more strongly associated with wheezing in the third year (or = . ; % ci, . to . ) than having one or more rsv-associated wheezing episodes during the first years of life (or = . ; % ci, . to . ). first-year wheezing associated with hrv was the strongest predictor for third-year wheeze (or = . ). the pattern of viral respiratory tract infection in the first years was different for children with or without asthma at the age of years (fig. - ) . asthma at years of age was strongly associated with hrv-associated wheeze in the first years of life (or = . ; % ci, . to . ). the frequency of hrv-induced wheezing episodes increased in the first years of life for children diagnosed with asthma by age , whereas for children without asthma, hrv-associated wheezing episodes declined in the first years (see fig. - ) . almost % of children with hrv-associated wheezing episodes in year had been diagnosed with asthma by the age of years, and hrv-associated wheeze was a more robust predictor for subsequent asthma than atopic sensitization to aeroallergens. asthma at years of age was also associated with rsv-associated wheezing episodes in the first years (or = . ; % ci, . to . ). the likelihood of asthma by age years being associated with rsv-induced wheeze in the first years of life was increased these findings are provocative and indicate another potential risk factor for asthma and allergic disease, one that is increasingly prevalent and amenable to intervention. a better understanding of the mechanisms and potential role of intervention in the primary and secondary prevention of disease is needed. respiratory infections are common in the first years of life, and they provoke wheezing in children with or without asthma. less certain is whether viral or other respiratory infections have a direct role in the pathogenesis of asthma or they merely reveal that a child is predisposed to asthma. investigation of the association of viral infection and asthma has been limited by available technology, with culture, serology, and antigen detection having % to % detection rates. the newer molecular technologies have improved the rates of viral detection up to about % and have revealed the importance of previously unknown viruses, such as human rhinovirus c (hrv-c). lower respiratory tract infections in children, which are caused by hrvs, respiratory syncytial virus (rsv), parainfluenza viruses, and other pathogens, are universal in childhood. a community-based study in tecumseh, michigan, estimated that children experience, on average, . lower respiratory tract infections in the first year of life and . such infections between and years of age. another cohort study of respiratory illnesses from birth through months in albuquerque, new mexico, adapted a surveillance system similar to the one used in tecumseh and found comparable incidence rates from through . the incidence of severe episodes of viral respiratory infections was captured in another study using surveillance through a pediatric group practice. this study showed that % of children were affected in the first year of life, that % had annual occurrences by age years, and that % of children to years old experienced annual episodes of infection. follow-up studies of children with a history of hospitalization for respiratory infections suggest that these illnesses may predispose to the development of asthma. in several studies, children with past hospitalizations tended to have abnormal lung function that was indicative of airflow obstruction, including hyperinflation, increased respiratory resistance, and reduced spirometric flow rates. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in children with past hospitalizations, increased airway reactivity occurred after assessment by exercise, cold air inhalation, methacholine, or histamine inhalation challenge. [ ] [ ] [ ] infants hospitalized with rsv-associated bronchiolitis are more likely to wheeze and develop asthma later in childhood. a study of swedish children found that those who were hospitalized with rsv bronchiolitis in infancy were almost nine times more likely to have physician-diagnosed asthma at age years than those without infection. being hospitalized with rsv bronchiolitis in infancy was an independent risk factor for current asthma and recurring wheezing (or = . ; % ci, . to . ). henderson and colleagues described the relationship of hospitalization for rsv bronchiolitis in infancy and asthma in a population-based birth cohort study of more than children from the united kingdom. hospitalization for rsv bronchiolitis was associated with physician-diagnosed asthma at age years (or = . ; % ci, . to . ) only among nonatopic children. no association was observed for children with atopy type (hrv- ) replicates more readily in the airway epithelial cells of people with asthma, and the airway epithelial cells are more likely to lyse and have greatly impaired interferon-λ (ifn-λ) and ifn-β responses. van der zalm and coworkers reported that increased neonatal airway resistance was related to an increased likelihood of hrv-associated wheeze in the first year of life (or = . ; % ci, . to . ). hrv was originally classified as serotypes hrv-a and hrv-b, but in , a novel hrv designated hrv-c was identified using reverse transcription-polymerase chain reaction (rt-pcr). hrv-c has been implicated in the natural history of wheezing disease and asthma, and it appears to have prognostic importance. in a prospective, population-based study of children younger than years of age who were hospitalized in two u.s. counties with acute respiratory illness or fever, hrv was detected in %, and hrv-c was isolated slightly more than % of them. children from whom hrv-c was isolated were significantly more likely than those with hrv-a or hrv-b to have underlying high-risk conditions such as asthma (or = . ; % ci, . to . ). in australia, hrv serotypes were isolated from % of children to years old who presented to the hospital with acute asthma. hrv-c was isolated from % of the children, and these children had higher asthma severity scores than those infected with hrv-a or hrv-b. in a study of children hospitalized in hong kong, hrvs were isolated from % of children admitted because of acute asthma and from % of control, nonatopic children hospitalized with nonasthma respiratory conditions. hrv-c was isolated from % of the children with acute asthma and % of the controls, and children with hrv-c were more likely to require supplemental oxygen. these studies implicate hrv-c in most episodes of acute asthma requiring hospital attention. hrv-c appears to be more virulent than other hrv serotypes, particularly in children with atopic sensitization. although the major focus has been on viral respiratory tract infection, asymptomatic early-life bacterial airway colonization has also been associated with childhood wheeze and asthma. in the copenhagen prospective study of asthma in childhood, neonates at high familial risk for asthma had their hypopharyngeal regions sampled at month of age, and the children were then followed up to years of age. neonatal colonization of the hypopharyngeal region by streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis (but not staphylococcus aureus) in isolation or in combination was associated with increased likelihood of subsequent wheeze, hospitalization with wheeze, and asthma. hypopharyngeal colonization at year of age was not associated with neonatal colonization or the development of wheeze or asthma. although has been postulated that early-life bacterial colonization induced neutrophilic airway inflammation with consequent wheeze and asthma, it also has been suggested that neonatal airway colonization by these bacteria reflects defective early-life innate immune responses that predispose to asthma. asthma-like symptoms, especially in young children, often are treated with antibiotics, and an association has been observed between the use of these drugs and the risk of asthma. the simultaneously increased use of antibiotics in children and the increasing prevalence of asthma in developed countries has led to the hypothesis (consistent with the hygiene hypothesis) that antibiotic use may contribute to asthma by altering the normal colonization of gut flora in infants and increasing the only for children who had also had hrv-associated wheezing episodes. measurement of lung function in the coast cohort at age years demonstrated that hrv-associated wheezing episodes in the first years of life were associated with reduced lung function: fev of % of predicted for those with hrvassociated wheeze versus % for no hrv-associated wheeze (p < . ). similar differences were found for absolute fev , forced expiratory volume in . second (fev . ), and forced expiratory flow determined over the middle % of a patient's expired volume (fef ). lung function at age was not associated with the frequency of hrv-associated wheeze nor with rsv-associated wheeze. although studies such as coast demonstrate that hrv respiratory infection is prognostically more important than rsv infection for subsequent asthma, whether virus-associated wheezing episodes (particularly hrv) contribute to the pathogenesis of asthma or are merely manifestations of infection in children predisposed to asthma remains an unanswered question. there is evidence supporting the concept that children predisposed to asthma have lung function and airway epithelial abnormalities from very early in life that increase the likelihood of virus-associated wheezing episodes. [ ] [ ] [ ] human rhinovirus cross-sectional studies. in one of the earliest reports, northway and coworkers considered the first possibility-that asthma is a long-term consequence of bronchopulmonary dysplasia (bpd). bpd is a syndrome of chronic lung disease in premature infants who are mechanically ventilated for at least week as a treatment for rds. the clinical diagnosis requires the symptoms of persistent respiratory distress during infancy, dependence on supplemental oxygen, and abnormal chest radiographs. northway and colleagues then studied adolescents and young adults born between and who had bpd in infancy and compared their long-term pulmonary outcomes with those of two control groups. they found that most subjects with a history of bpd in infancy had pulmonary dysfunction. moreover, the increase in airway reactivity was not associated with a more frequent family history of asthma in this sample or with an increased prevalence of atopy. these findings suggest that lung injury resulting from mechanical ventilation of premature infants has a role in the pathogenesis of persistent pulmonary dysfunction that is similar to asthma. bertrand and associates investigated the role of rds in prematurity in the pathogenesis of airway hyperresponsiveness (ahr) in subjects who did not have bpd as infants. the group with a history of rds had evidence of more hyperinflation and airway obstruction compared with controls. however, results from the histamine challenge to determine ahr and familial aggregation of ahr were inconclusive. the incidence of airway reactivity was elevated among cases and controls and among the mothers and siblings of cases and controls. the investigators suggest that the elevated incidence of ahr among mothers of both groups supports the hypothesis that there may be an association between the onset of premature labor and airway reactivity. because no comparison group was established for mothers of term children, however, this assertion cannot be affirmed from the study. some researchers have investigated the effect of very low birth weight (vlbw < g) and bpd on asthma development in birth cohorts. [ ] [ ] [ ] [ ] [ ] [ ] [ ] children with vlbw were followed for years as part of the newborn lung project conducted in wisconsin and iowa. , results at age years did not show a consistent association between asthma and bpd. children with diagnosed bpd and children with radiographically identified bpd had about a threefold and twofold increase, respectively, in the risk of bronchodilator use up to age years, adjusted for birth weight, gestational age, gender, race, and neonatal center. among children with bpd, the prevalence of ever having asthma at age years did not show a difference by the period of birth. however, the prevalence of wheezing in the last year at years of age decreased from % to % over time. as the researchers observed, this finding could have resulted from the introduction of surfactant therapy as a bpd treatment. prematurity as a risk factor for asthma has been explored in cross-sectional studies. [ ] [ ] [ ] [ ] [ ] [ ] a significant association between current asthma prevalence and premature girls was observed in a study of schoolchildren. significantly more premature children had a family history of asthma than did term children, and this association was stronger among children who required mechanical ventilation as premature infants. another german study of schoolchildren did not show an association between former or current asthma and low birth weight (lbw < g) among premature children. however, bronchial hyperresponsiveness was significantly increased in children born at atopic, helper t cell type (th ) immune responses. , in support of this hypothesis, humans exposed to stable and farm environments, which are rich in microbes, show significantly reduced levels of asthma and atopic disease compared with those in other rural or nonrural environments. other studies have shown that the different proportion of aerobic and anaerobic gut flora in children from sweden compared with estonia parallels the difference in atopy incidence between these populations. , animal studies also support the hypothesis. mice given oral antibiotics had altered intestinal flora and impaired helper t cell type (th ) immune responses. epidemiologic studies of asthma and allergic disease in relation to antibiotic use are beset by biases, including reverse causality (i.e., asthma leads to more common prescription of antibiotics) and confounding by indication (i.e. respiratory infections leading to antibiotic use may be implicated in the development of asthma). to illustrate this problem, in a carefully conducted tucson birth cohort study, information on illness, antibiotic use, and physician visits was ascertained on seven occasions in the first months of life and correlated with the development of asthma and allergic disease up to the age of years. a significant association between the number of early-life courses of antibiotics and asthma was reported. the number of physician visits was associated with the number of antibiotic courses and with asthma. however, after adjustment for the number of physician visits, antibiotic use was not associated with asthma, and it was concluded that any association between early-life antibiotic use and asthma was an artifact of the number of physician visits for illness, which was strongly associated with antibiotic use and risk of asthma. two systematic reviews have provided insight into the possible causative association between early-life antibiotic use and asthma and allergic disease. a systematic review of studies that have related antibiotic exposure during pregnancy or in the first year of life with risk of childhood asthma identified relevant studies. antibiotic use in the first year of life was associated with an increased likelihood of childhood asthma (or = . ; % ci, . to . ). stratified analysis indicated that retrospective studies reported the strongest associations (or = . ; % ci, . to . ) compared with database and prospective studies (or = . ; % ci, . to . ) . studies that addressed potential biases by adjusting for respiratory infections reported the weakest associations (or = . ; % ci, . to . ). a second systematic review focusing on longitudinal studies identified studies, and a meta-analysis indicated that antibiotic use was associated with subsequent wheeze or asthma (or = . ; % ci, . to . ). however, after eliminating nine studies with a high risk of bias, the magnitude of the association was reduced (or = . ; % ci, . to . ). both systematic reviews concluded that there might be a weak link between antibiotic use and subsequent asthma and that biases had exaggerated the strength of any association that might exist. premature birth has been associated with the development of symptoms consistent with asthma and other long-term pulmonary sequelae in a number of studies. the cause of the sequelae is uncertain. the pulmonary injury may be acquired during mechanical ventilation of preterm infants with respiratory distress syndrome (rds), from the rds itself, or from some other facet of prematurity. prematurity has been examined as a risk factor for asthma in cohort studies of affected children and in plants (e.g., grain dust, flour, latex, castor bean, green coffee bean), enzymes (e.g., subtilisin from bacillus subtilis, papain, fungal amylase), wood dust or barks (e.g., western red cedar, oak, reactive dyes), drugs (e.g., penicillin, methyldopa), metals (e.g., halogenated platinum salts, cobalt), and others such as oil mists. they have been classified according to possible pathogenetic mechanisms: high-molecular-weight agents that induce specific ige antibodies; low-molecular-weight substances, such as isocyanates, for which underlying mechanisms are largely unknown; and irritant gases, fumes, and chemicals that induce occupational asthma by nonimmunologic mechanisms. more extensive coverage of these agents and the topic is available elsewhere. , other causes of occupational asthma have been identified through clinical reports, epidemiologic investigations, and population studies. jaakkola and colleagues conducted a casecontrol study in finland. risk for asthma was found to be increased for several occupational groups, including some for which occupational asthma had not been previously reported, such as being a male or female waiter. le moual and coworkers explored associations for occupation and occupational exposures with asthma in , participants in a french survey conducted in . several jobs were associated with an increased risk of asthma of about %. a similar analysis was reported for the united states based on the nhanes iii. several studies provide estimates of the overall importance of occupational asthma. kogevinas and colleagues analyzed data from more than , young adults participating in the ecrhs. an estimated . % of asthma was attributed to occupation, with asthma defined by asthma symptoms or use of medication and assessed by questionnaire. when asthma was defined by questionnaire responses and bronchial hyperresponsiveness, the attributable risk estimated for occupation increased to . %. among members of a u.s. health maintenance organization, one third of persons identified as having new or recurrent asthma were classified as having a potential association with work as the basis for asthma. blanc and toren conducted a meta-analysis of studies on occupational asthma from to mid- . the median attributable risk estimate for occupational asthma was % for all studies identified. when the study quality was taken into account and analyses were limited to those of higher quality, the estimate was %. these estimates included new-onset asthma and reactivation of preexisting asthma. outdoor air pollutants can be classified by origin as natural or manmade. among the naturally occurring air pollutants are particulate matter (including bioaerosols), volatile organic compounds, and ozone. for asthma, the key manmade pollutants result from combustion of fossil fuels in cars, power plants, heating devices, and industrial point sources and from emissions of chemicals from manufacturing facilities, storage tanks, and accidental releases. in the united states, air pollutants have been categorized on the basis of their regulation under the clean air act as criteria pollutants (e.g., lead, nitrogen dioxide [no ], sulfur dioxide [so ], particulate matter [pm], ozone [o ], carbon monoxide [co]) and as air toxics, a specified listing of chemicals that includes some irritants relevant to asthma. these pollutants are a concern throughout the world's polluted cities and regions. many cities and smaller towns and term with lbw compared with children born with normal birth weight, with values adjusted for height, gender, and age. a study conducted as part of the ecrhs examined birth characteristics and asthma symptoms in young adults from norway. the researchers observed a significant decrease in asthma symptoms per -g increase in birth weight, adjusted for gestational age, length at birth, parity, maternal age, gender, adult height, hay fever, and current smoking habits. race and socioeconomic status may be determinants of prematurity and asthma. to test the hypothesis that prematurity was a risk factor for asthma independent of race or socioeconomic status, oliveti and colleagues performed a case-control study using a population restricted to african-american children from impoverished inner-city census tracts in cleveland, ohio. their findings confirmed previous findings with regard to prematurity and lbw. asthmatic children had significantly lower birth weights and gestational ages than nonasthmatic children and were more likely to have required positive-pressure ventilation (ppv) after birth. the risk of asthma was increased more than three times for children receiving ppv after birth. however, the increased risk of asthma due to lbw and prematurity was not significant when maternal history of asthma, bronchiolitis, lack of prenatal care, low maternal weight gain, and ppv were considered simultaneously. this suggests that lung injury and perhaps mechanical ventilation lead to an asthma-like syndrome, rather than lbw and prematurity directly. researchers have examined the lung function of preterm children over time. koumbourlis and associates followed preterm children with chronic lung disease, including bpd, from to years of age. the investigators observed improvements in the lung volumes of these patients throughout childhood and into adolescence, and these improvements were experienced by all children, regardless of the severity of the neonatal chronic lung disease. if patients had airway obstruction, it was primarily localized to the smaller airways, associated with ahr, and relatively fixed over time. two systematic reviews have investigated the association between prematurity and childhood asthma and wheezing outcomes. patelarou and colleagues identified nine studies that had reported on the association between adverse birth outcomes (e.g., premature, lbw, vlbw, fetal growth retardation) and early ( to years) childhood wheeze. they concluded that adverse birth outcomes were associated with wheezing in early life. similarly, a systematic review that identified studies reported that preterm (< weeks' gestation) was associated with an increased likelihood of childhood asthma (or = . ; % ci, . to . ). these results suggest that premature infants with or without neonatal respiratory disease may be at higher risk for asthma or a syndrome similar to asthma than term infants. however, the mechanistic pathways involved and the potential interactions with other asthma risk factors, such as viral respiratory infections and susceptibility genes, remain uncertain. occupational asthma is defined as variable airflow limitation or bronchial hyperresponsiveness due to exposure to a specific agent or conditions in a particular occupational setting but not to stimuli encountered outside the workplace. several hundred agents have been identified as causes of occupational asthma. , they include animal allergens (e.g., urine, dander), for children from east germany, where pollution originated from burning brown coal and industrial emissions. however, living in west germany was not an independent risk factor for asthma after adjustment for sensitivity to pollen, hdms, and cat allergens. another german study conducted from through obtained similar results. current asthma prevalence for children from munich was . %, compared with the prevalence for their counterparts from dresden of . %. significant differences in physician-diagnosed asthma prevalence were observed by comparing children in munich ( . %) and those in dresden, former east germany ( . %). a study enrolling children to years of age who were living in hong kong compared physician-diagnosed asthma prevalence in a high-pollution district and a low-pollution district. the researchers found that asthma prevalence was almost doubled in the high-pollution area compared with the low-pollution area. some studies have investigated the possible role of specific air pollutants in the development of asthma. in a cross-sectional study that was conducted as part of the isaac phase two and enrolled the same german children from dresden, an increase in estimated traffic-related exposure to benzene was associated with an increased prevalence in physician-diagnosed asthma after adjusting for potential confounders. however, this association reached statistical significance only when the home and school addresses used as the exposure indicators were combined. the prevalence of asthma was not associated with concentrations of so , no , and co. an increase in the exposure to air pollutants (except ozone) was associated with an increased prevalence of physician-diagnosed asthma in nonatopic children ( to years and to years old). this relationship was not observed in atopic children. another cross-sectional study evaluating the effects of general air pollution was conducted among , high school students in taiwan as part of the isaac. the researchers investigated the role of long-term exposure (i.e., annual average concentration) to air pollution and the prevalence of asthma. long-term exposure to total suspended particles, no , co, ozone, and airborne dust was associated with increased prevalence of asthma after adjusting for exercise, smoking, alcohol consumption, incense use, and environmental tobacco exposure. a similar study of , middle school students living in counties and cities in taiwan found a positive association between physician-diagnosed asthma prevalence and exposure to co and nitrogen oxides (no x ) when adjusted for age, history of atopic eczema, and parental education. baldi and coworkers reanalyzed data from a survey of children and , adults from seven french towns between and . they estimated a significant increase (or = . ; % ci, . to . ) in asthma prevalence per µg/m in the so -year-period annual mean after adjusting for age, education, and smoking status. the association remained significant when they restricted the analysis to adults reporting their first attack after moving to the study areas. they did not observe this relationship for children. these cross-sectional studies address the prevalence of asthma, which reflects the incidence and duration of the disease. if air pollution increases the duration of asthma, the prevalence would be increased, even without an effect on incidence. the clearest evidence of a causal association between outdoor air pollution and childhood asthma comes from cohort studies. villages in the developing world have the problem of smoke from biomass fuel use for indoor cooking and heating that is emitted outdoors. although it is accepted that exposure to outdoor air pollution can exacerbate existing asthma, - the role of outdoor air pollution in the development of childhood asthma is less well established. however, there is increasing evidence, especially from studies with a focus on exposure related to traffic within urban areas, that implicates outdoor air pollution in the development of childhood asthma [ ] [ ] [ ] and lung function. the outbreaks of acute asthma in barcelona illustrate the consequences of exposure to an airborne contaminant and the need to investigate asthma epidemics. during the s, a remarkable series of epidemics of asthma occurred in barcelona, a port city. careful analysis of one outbreak showed spatial clustering near the harbor, and an epidemiologic investigation showed a very strong association between unloading of soybeans at the harbor and occurrence of the epidemics. an antigen was identified in the soybeans that proved to be responsible for the outbreaks. the outbreaks were traced to releases of dust at a particular silo, and control measures were enacted. subsequently, a review of the historical record showed that there had been similar outbreaks of soybean asthma in new orleans. a large body of experimental and observational evidence links outdoor air pollution to exacerbation of asthma. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] compilations of the evidence can be found in the criteria documents prepared by the u.s. environmental protection agency (epa) for particulate matter and ozone. , human experimental studies have provided some insights, showing for example, that the oxidant pollutants nitrogen dioxide and ozone may enhance the effects of allergens, possibly by increasing the permeability of airways. , epidemiologic data, primarily coming from studies of panels of persons with asthma or of medical morbidity, have shown that the adverse effects of air pollution on asthma are relevant clinically and are significant from a public health perspective. there is uncertainty about the relative effects of specific pollutants compared with the overall toxicity of the air pollution mixture. gent and colleagues investigated the effect of exposure to ozone and particulate matter of . µm in diameter (pm . ) in a u.s. cohort study of asthmatic children. among children using maintenance medication, the level of ozone, but not pm . , was significantly associated with worsening of respiratory symptoms and an increase in rescue medication use. significant associations were not found for children not using maintenance medication. these findings suggest that children with asthma using maintenance medication are especially vulnerable to ozone, even after adjusting for exposure to pm . and at air pollution levels below the epa air quality standards. various lines of epidemiologic evidence continue to indicate a potential role of air pollution in the cause of asthma. crosssectional studies have investigated asthma prevalence and air pollution. after the unification of east and west germany, studies were conducted to compare respiratory diseases among children who had a relatively homogenous genetic background but had experienced exposures to air pollution at very different concentrations. [ ] [ ] [ ] [ ] in a study conducted between and , children to years old from munich (west) had a higher prevalence of physician-diagnosed asthma than those from leipzig and halle (east). current asthma prevalence among children living in west germany, an area with a greater amount of heavy road traffic, was . %, compared with . % also raised. the disparity between cross-sectional and prospective studies suggests that although the incidence of asthma among those living close to traffic is increased, it is not evident at a population level because of the small effect size and the lack of variation in the distance between home and traffic. cohort studies published since the comeap are consistent with its findings, but they also highlight a possible early-life effect and the importance of exposure while at school. the dutch prevention and incidence of asthma and mite allergy (piama) birth cohort study related symptom data prospectively collected annually from children up to the age of years to land-use regression estimates of individual no , pm . , and soot exposures at their birth addresses. pm . was associated with an increased annual incidence of asthma (or . ; % ci, . to . ), prevalence of asthma (or = . ; % ci, . to . ), and asthma symptoms (or = . ; % ci, . to . ). the associations between outcomes and no and soot exposures were similar, but there was a high correlation (r > . ) for pm . , no , and soot exposures. only % of the cohort were still living at the birth address at age years, and the associations between pollutants and outcomes were evident only in those who had not moved house; for the children who had moved from the birth addresses, the only significant association was between pm . and the prevalence of wheezing symptoms (or = . ; % ci, . to . ). the southern californian health study evaluated symptom-free children recruited in kindergarten or the first grade (≤ years old) from communities, each with continuous ambient ozone, no , pm . , and pm measurement. the incidence of asthma in the subsequent years was determined by annual questionnaires and correlated with individualized estimates of traffic-related pollution at home and at school. the incidence of asthma was increased by nonfreeway traffic-related pollution at home (hazard ratio [hr] = . ; % ci, . to . ) and at school (hr = . ; % ci, . to . ) . although the balance of evidence suggests an association between outdoor air pollution and the development of asthma in some individuals who live near busy roads, there does not appear to be an association between air pollution and the development of asthma at the population level. moreover, the welldocumented increase in asthma prevalence in the latter decades of the twentieth century cannot be readily explained by changes in levels of the major combustion pollutants. the emerging association between traffic-related emissions and asthma requires further investigation. in the home and other indoor environments, children and adults inhale diverse pollutants that may be associated with the risk for asthma. , they include combustion-source emissions from cooking stoves and ovens, space heaters fueled by gas or kerosene, wood-burning stoves or fireplaces, and tobacco smoking; volatile and semivolatile organic compounds released from household products, furnishings, and other sources; and allergens from insects, molds, mites, rodents, and pets. , many of these pollutants can be present in higher concentrations indoors than outdoors, providing a rationale for studies that have examined indoor pollutants as factors that may cause or exacerbate asthma. for example, in a prospective cohort study of inner-city u.s. children with asthma, indoor no and pm were associated with asthma symptoms. the associations were independent of each other and of outdoor the traffic-related air pollution and childhood asthma (trapca) study is a birth cohort study of children from the netherlands, germany, and sweden that is funded by the european union. preliminary results from the german children followed for their first years of life showed a % ( % ci, . to . ) increase in the risk of asthmatic, spastic, or obstructive bronchitis for those living close to major roads (< m) compared with children farther away. a cohort study of almost children between the ages of and years, who lived in nine communities surveyed in the california children's health study and four other communities, was started in to evaluate characteristics that might increase children's susceptibilities to the effects of traffic-related pollution. preliminary results showed that living within m of a major road was associated with an increased risk of physician-diagnosed asthma (or . ; % ci, . to . ), prevalent asthma (or = . ; % ci, . to . ), and wheeze (or = . ; % ci, . to . ). among long-term residents (i.e., living in the same home since the child was years old or younger) with no parental history of asthma, an increased risk of physician-diagnosed asthma (or = . ; % ci, . to . ), prevalent asthma (or = . ; % ci, . to . ), and wheeze (or = . ; % ci, . to . ) was associated with living within m of a major road. increased risk was not associated with the exposure for children with a parental history of asthma and for short-term residents. the adventist health study on smog (ahsmog) is a prospective cohort study that enrolled more than nonsmoking adults ( to years old) living in california in . in the first years of follow-up, abbey and colleagues examined incident asthma cases in relation to pm and found a % increased risk of asthma for a hr/yr exposure to concentrations of pm that exceeded µg/m . a later report on the ahsmog participants used the - -hour mean ozone concentration as the exposure and found that the risk of developing asthma doubled per parts per billion increase for males but not in females after adjusting for age, education, respiratory infection before age years, and smoking status. a systematic review commissioned by the u.k. committee on the medical effects of air pollution (comeap) was established to investigate whether outdoor air pollution causes asthma. this review identified cross-sectional studies relating asthma prevalence in more than four cities to quantitative pollution measures; the number of cities ranged from to and covered europe, north america, and asia. a metaanalysis revealed no significant associations between no , pm , or so and period prevalence of wheeze and lifetime prevalence of asthma. the review also identified studies of birth cohorts and studies of cohorts recruited during child or adulthood. in these studies, exposures were individualized by modeling, usually to the individual's home address. in contrast to the cross-sectional studies, meta-analysis revealed associations between no and the incidence of asthma (or = . ; % ci, . to . ; studies) and between pm . and the incidence of asthma (or = . ; % ci, . to . ; studies). the comeap systematic review concluded that the evidence from the cohort studies is consistent with a significant increase in the incidence of asthma associated with no and pm from traffic sources. the possibilities of air pollution aggravating existing subclinical asthma and residual confounding by factors associated with asthma and residential proximity to traffic were assessed early indoor allergen exposure and physician-diagnosed asthma or wheeze and did not find an association. they concluded that their results did not support the hypothesis that allergen exposure causes asthma. prospective cohort studies have studied the relationship between exposure to mold and the risk of asthma. a study of finnish children to years old used parents' reports of mold and dampness as a surrogate for exposure to aeroallergens in the home. after years of follow-up, exposure to mold was found to be an independent risk factor for asthma among finnish children. the incidence of physician-diagnosed asthma was double for children in homes with reported mold odor compared with those that did not. jaakkola and jaakkola reviewed the literature on indoor molds and asthma, and they concluded that exposure to molds at home increases the risk of asthma among adults and that exposure to molds at work increases the risk of wheezing. they observed that exposure to indoor molds increases the severity of asthma and that removing the source relieves or eliminates symptoms and signs of asthma. sensitization to mold has been linked to the presence, persistence, and severity of asthma. , a review of housing interventions designed to improve outcomes concluded that asthma symptoms could be reduced by removing moldy items and eliminating leaks and other moisture sources in homes. intervention studies with avoidance of aeroallergens and food allergens have not consistently found a reduction of asthma risk among children. the canadian childhood asthma primary prevention study included high-risk children who were randomized to intervention (i.e., avoidance of hdm by use of mattress covers and acaricides, pets, and passive smoking and encouragement of breastfeeding with delayed introduction of solid foods) or to control groups before birth. for children at years of age, the prevalence of physician-diagnosed asthma was significantly lower for the intervention group ( %) than for the control group ( %). another intervention study of a birth cohort of high-risk children living on the isle of wight assessed asthma (i.e., wheeze and bronchial hyperresponsiveness) prevalence at age years and found that the asthma risk was ninefold higher for the control group than the intervention group. intervention included breastfeeding by a mother on a low-allergen diet or giving a hydrolyzed formula and reducing hdm exposure with an acaricide and mattress covers. however, the australian childhood asthma prevention study, which included highrisk children randomized to an hdm avoidance intervention group or control group, did not find a significant reduction in the prevalence of current asthma at age years for the intervention group compared with the control group. a systematic review and meta-analysis of prospective birth cohort studies evaluating the effects of allergen (i.e., hdm or dietary) avoidance during pregnancy concluded that early-life allergen avoidance in isolation does not reduce the likelihood of asthma in children at age years (or = . ; % ci, . to . ). however, multifaceted antenatal intervention that combines breastfeeding with allergen avoidance and maternal smoking cessation does reduce the likelihood of asthma in children at age years (or = . ; % ci, . to . ). exposure to tobacco smoke has serious adverse effects on the respiratory tract. perhaps because of the sensitivity of the concentrations of the pollutant. a full examination of this literature is beyond the scope of this chapter, but reviews of indoor air pollution are available. whether these exposures by themselves, in the absence of underlying genetic susceptibility, can cause asthma is uncertain. however, mounting evidence indicates that maternal smoking is associated with an increased risk for asthma in offspring and later exacerbations of asthma (see "involuntary or passive smoking") and that levels of allergen exposure are associated with the incidence of asthma and wheezing. however, there have been only limited investigations of indoor air pollution and the incidence of asthma linked to risk factors other than passive exposure to tobacco smoke. an institute of medicine committee reviewed the evidence on indoor air pollution and childhood asthma and derived conclusions regarding causation and exacerbation. this topic also has been reviewed elsewhere. , several investigations have addressed the prevalence of asthma and exposure to nitrogen oxides from cooking stoves. homes with natural gas-fueled or propane-fueled cooking stoves tend to have no levels substantially above those of homes with electric stoves. some investigations indicate a general increased risk of respiratory symptoms, including wheezing, in households with gas stoves, but the data are inconsistent and not indicative of increased asthma incidence caused by nitrogen oxides. , the myriad exposures to volatile and semivolatile organic compounds that can occur in homes and other locales have been investigated as risk factors for childhood asthma. although many cross-sectional studies report an association between volatile organic compound exposure and asthma in children , and adults, , these studies cannot establish causality and are beset by the problem of reverse causality, whereby parents modify their houses (e.g., laminate flooring) as a consequence of their children developing asthma. cohort studies suggest that maternal volatile organic compound exposure during pregnancy can influence the development of childhood allergic disease. this is an area of ongoing research because of the potential for intervention by behavioral modification and low volatile organic compound technology. studies of indoor allergens have largely focused on the status of children with asthma in relation to levels of allergen rather than considering the levels of allergens as predictors of asthma. a prospective cohort study conducted in the united kingdom found levels of hdms in the home to predict later development of asthma, and children with higher levels of hdm antigen in their homes tended to wheeze at a younger age. the german multicentre allergy study followed children from birth to years of age and found that sensitization to perennial allergens such as hdms, cat hair, and dog hair that developed before years of age was associated with a loss of lung function at school age. a u.s. study of children indicated that exposure to two or more dogs or cats in the first year of life might reduce subsequent allergic sensitization risk to multiple allergens during childhood. not all studies support the conclusion that allergen exposure causes asthma. a british cohort study did not find a significant association between levels of hdm exposure and sensitization or wheeze. results from a german birth cohort of children followed until age years showed a strong association between sensitivity to hdm allergens or cat allergens and wheezing from years of age. however, the investigators also during pregnancy has also been associated with increased in vitro cord blood mononuclear cell proliferative and cytokine responses after stimulation with allergens. , there is extensive literature on the relationship between passive smoking and childhood wheeze and asthma. a systematic review identified relevant prospective cohort studies. exposure to maternal (prenatal and postnatal), paternal, and household sources of cigarette smoke was associated with an increased likelihood of children wheezing up to the age of years. the strongest associations for childhood wheeze were for postnatal exposure to maternal cigarette smoking: wheeze at years or younger (or = . ; % ci, . to . ), to years (or = . ; % ci, . to . ), and to years (or = . ; % ci, . to . ). the associations between exposure to maternal, paternal and household cigarette smoke and childhood asthma were not as strong as for wheeze, but they were most noticeable for maternal smoking during pregnancy: childhood asthma at years or younger (or = . ; % ci, . to . ) and to years (or = . ; % ci, . to . ). paternal smoking was associated with an increase in childhood asthma between and years, and household smoking was associated with an increase in childhood asthma after the age of years. the children's health study based in california reported a transgenerational association, suggesting that exposure to cigarette smoke in utero may have epigenetic effects. in a nested case-control study of children at years of age ( with asthma and controls), the likelihood of childhood asthma was increased if the mother (or = . ; % ci, . to . ) or the maternal grandmother (or = . ; % ci, . to . ) smoked during pregnancy. if the mother and grandmother smoked during pregnancy, the likelihood of childhood asthma was increased further (or = . ; % ci, . to . ). although allergic rhinitis is common, few epidemiologic studies have focused on this disease. the most frequently cited risk factors include increasing age, atopy, and high socioeconomic status. parental history is positively associated with the development of allergic rhinitis in offspring. in the tucson birth cohort study, a maternal history of physician-diagnosed allergy was significantly associated with a diagnosis of rhinitis by age years (or = . ; % ci, . to . ). perinatal and infant risk factors have been examined. for example, younger gestational age at birth has been associated with a decreased risk of allergic rhinitis. , some researchers have postulated that early-life exposures to microbes may modulate risk of allergic rhinitis, and this hypothesis has been supported by the observations that birth by cesarean section is a risk factor for allergic rhinitis, as is reduced diversity of the intestinal microbiota in infancy. other risk factors under investigation include genetics, early-life exposure to infections, acetaminophen use, oral contraceptive use, and indoor and outdoor air pollution exposure. risk factors for eczema include gender, race or ethnicity, family history, early-life antibiotic use, environmental exposures, and dietary factors, including breastfeeding, timing of the introduction of solids, and inclusion of probiotics. family history of asthmatic lung to cigarette smoke, young smokers tend to have somewhat greater lung function and less underlying airway responsiveness than nonsmokers-a phenomenon sometimes referred to as the healthy smoker effect. nonetheless, substantial data show that active smoking increases nonspecific responsiveness of the airways, perhaps by inducing inflammation or by narrowing baseline airway caliber in older people. smokers also tend to report wheezing more frequently than nonsmokers, and wheezing tends to decline after cessation of smoking. increased airway responsiveness in active smokers also tends to abate after smoking cessation. , a systematic review of studies exploring the temporal association between active smoking and asthma reported that most studies indicated that people who smoked were at increased risk for asthma. these studies evaluated diverse sample populations and used different methods, and the review highlighted the potential for residual confounding by health behaviors (e.g., physical exercise). the review concluded that although active smoking might be a risk factor for asthma, the evidence was insufficient to conclusively state whether smoking was a causal or proxy risk factor for asthma. the nonsmoking child is exposed to second-hand smoke, a name given to the mixture of sidestream smoke released by a burning cigarette and the mainstream smoke exhaled into the air by the smoker. this mixture has also been called environmental tobacco smoke. smoking adds respirable particles and irritant gases to indoor air, and it represents one of the major sources of fine particles in the air of u.s. homes. exposure of children to particles and gases in tobacco smoke has been documented by measuring personal exposures and using biomarkers that indicate the levels of tobacco smoke components absorbed into the body. cotinine, a major metabolite of nicotine, has been extensively investigated in children in relation to parental smoking. compared with children living in households in which there is no smoking, children living with smokers tend to have substantially higher cotinine levels. , in the past, exposure to second-hand smoke was widespread. almost all participants, including nonsmokers, in the - nhanes iii had detectable serum cotinine levels. ten years later, nhanes iv showed a dramatic reduction in cotinine levels, a trend that has continued. exposure to second-hand smoke contributes to both the causation and the exacerbation of asthma. first, passive smoking may increase the risk of more severe lower respiratory tract infections during the early years of life. second, the direct toxic effects of second-hand smoke may induce and maintain the heightened nonspecific responsiveness of airways found in asthmatic children. third, many children have secondhand smoke exposure during gestation and after birth. substantial evidence suggests that in utero exposure to tobacco smoke components affects fetal airway and immune system development. young and associates assessed nonspecific airway responsiveness using a histamine challenge for normal infants at a mean age of . weeks. even at this young age, parental smoking and a family history of asthma were associated with an increased level of airway responsiveness. in a similar prospective investigation, hanrahan and colleagues found that children whose mothers smoked during pregnancy had a lower level of airway function soon after birth. maternal smoking later epidemiologic studies provided a deeper understanding of the physiologic consequences of having childhood asthma and indicated that the lungs of these children might already have heightened airway responsiveness at birth. birth cohort studies that include indices of ventilatory function and airway responsiveness during the first weeks of life indicate that infants at risk for asthma because of a parental history of asthma and atopy already have heightened responsiveness to a challenge. the tucson study clarified the early natural history of wheezing. , martinez and colleagues described the natural history of wheezing beginning before years of age and found that some children had only transient early wheezing. children who continued to wheeze up to years of age were more likely to have mothers with a history of asthma and to have an elevated serum ige levels, suggesting that the early wheezing represented asthma. children whose wheezing did not persist had diminished airway function in early life but did not tend to have mothers with asthma or elevated ige levels. the pattern of persistence of wheezing during childhood and into adulthood was similar in a smaller cohort study of children in england, who were followed from birth to age years. in this highrisk cohort, early wheezing was not likely to persist, but wheezing at years of age did tend to persist. the results of these studies imply that clinicians should be cautious in labeling all early childhood illnesses with wheezing as asthma, because some children are predisposed to wheeze with respiratory infections because of reduced airway function. population-based groups of children have been followed over time in prospective cohort studies (table - ) . because most of these studies have drawn participants from defined populations, there is less potential for bias by the selection process, and the children with asthma are more likely to be representative. information collected from childhood to early adulthood is available from several investigations, including two particularly large studies involving lengthy follow-up: the cohort study in australia and the birth cohort study in the united kingdom. , findings of a number of smaller studies have been similar (see table - ). one of the first studies using a birth cohort design was conducted in australia, initially by williams and mcnicol. , [ ] [ ] [ ] on enrollment in , the children were years of age, and after years of follow-up, they were years old. [ ] [ ] [ ] wheezing tended to track over time, but % were no longer wheezing at years of age, and only % had wheezing at least weekly. those with more severe wheezing at age years tended to have a lower level of lung function tested by spirometry and to have a higher degree of airway responsiveness to a methacholine challenge. over time, some improved, but an approximately equal proportion worsened. at age years, % of the group with wheezy bronchitis at baseline was free of wheeze, and only % of this group had persistent asthma. symptoms continued in % of the original asthma group and in % of the severe asthma group. almost one half of the severe asthma group continued to have persistent asthma at age years. those with severe asthma had suffered a loss in lung function by years of age, but this loss did not progress in adulthood. children with milder symptoms did not have a significant loss of lung function. in another large, long-term study, members of the birth cohort in the united kingdom were followed up to age years. , , parents were interviewed when the participants eczema has been identified as a risk factor for eczema in several studies, pointing to genetic determinants of eczema. loss-offunction mutations in the filaggrin gene (flg), which encodes a protein critical to skin barrier function, have been directly linked to eczema, and approximately % of people heterozygous for these mutations develop eczema. black and asian race or ethnicity is a risk factor, along with male gender, , although isaac phase three found that worldwide, boys were less likely to have eczema than girls. earlylife exposure to endotoxin appears to protect against the development of eczema, as reported in several studies. , dietary factors, including breastfeeding, infant formulas, timing of solid food introduction, and supplementation with probiotics, have been studied. neither breastfeeding nor timing of solid food introduction has been associated with protection against eczema. [ ] [ ] [ ] [ ] [ ] [ ] evidence suggests that hydrolyzed infant formulas and supplementation with probiotics may afford some protection against eczema, , but study results are mixed, and infection by the probiotic organism has been reported in infants receiving probiotic supplementation. established risk factors for food allergy include male gender for children, eczema, and an atopic family history. [ ] [ ] [ ] other possible risk factors are diet and feeding practices during early childhood. controversy exists about whether early allergen introduction or allergen avoidance may predispose to the development of food allergy. the natural history of asthma is a concern for affected children, their parents, the clinicians providing care, and researchers. parents ask whether the child will outgrow asthma, and clinicians should be able to answer this question. researchers have studied the natural history of asthma and searched for factors that determine prognosis. during adulthood, the former asthmatic child may be exposed to environmental agents, including cigarette smoke, which may adversely affect respiratory health. childhood asthma has been postulated to increase the likely adverse effects of these exposures and other long-term consequences, such as persistent physiologic impairment from airway remodeling. , initial information on the natural history of childhood asthma largely came from cohort studies of children attending general practices or clinics. , these studies, some dating to the s, were a principal source of data on the natural history of asthma until population-based investigations were implemented beginning in the s. these early studies provided evidence of waning of clinical symptoms over time in a substantial proportion of children with asthma. however, most children tended to remain symptomatic. interpretation of these data is constrained by differences between past and current therapeutic approaches, possible lack of representativeness of children receiving care at a particular clinical facility, and by diversity of the research methods. these studies drew the participants from general practices and clinics, and presumably, more severe asthma was represented. nevertheless, they provide evidence that the prognosis is favorable for some children with asthma, even in an era antedating contemporary therapeutic approaches. bronchial challenge testing, and allergy testing. of the participants with complete data for the follow-up period, . % had persistent wheezing into adulthood, and only . % never reported wheezing. the remainder had various patterns of intermittent wheezing. predictors of persistent wheezing included sensitization to hdms, female sex, and smoking at age years. pulmonary function was reduced in those with persistent wheezing. evaluation of the natural history of asthma in adults is complicated by the occurrence of copd and the potential difficulty of separating copd from asthma. in adults, asthma includes disease originating in childhood and following its natural course into adulthood and asthma developing during the adult years. these natural histories have not been carefully delineated, although the lengthier studies of childhood asthma can provide information on its course into adulthood. there is less information on asthma in adulthood that is comparable to that on childhood asthma, such as the longitudinal picture of symptoms and clinical status. however, the effect of having asthma on the decline of lung function has been assessed, and there is limited information on the development of irreversible airflow obstruction in persons with asthma (table - ). the evidence on asthma and change in lung function over time is inconsistent with some studies showing were , , and years of age, and the participants themselves were interviewed at age and years. asthma tended to remit over time; of the children with a report of asthma or wheezy bronchitis before years of age, only % had wheezing in the last year at age years, although this figure increased to % at age years. lung function was evaluated in a sample of of the participants with a history of asthma or wheezy bronchitis and controls. for those not reporting wheezing at age years, lung function was only slightly reduced compared with controls. for those with wheezing, fev was reduced by approximately % compared with controls. similar results were found in a follow-up study of dutch individuals. subjects were extensively tested as children years earlier and reexamined as adults. the data revealed that % of persons were no longer considered asthmatic, % had an fev greater than % of predicted, % were no longer bronchial hyperresponsive, and % did not report asthmatic symptoms. results of these studies support the hypothesis that early intervention in mild asthma may lead to improved outcomes. in a longitudinal, population-based, cohort study carried out in dunedin, new zealand, children were enrolled, and a substantial proportion was followed to age years with repeated assessment by questionnaires, lung function testing, in the copd group and a -ml loss in the intermediate group. the balance of the evidence indicates that a diagnosis of asthma is associated with an increased rate of fev decline (see table - ). perhaps reflecting this excess decline, many elderly persons with asthma have fixed airflow obstruction. there are few studies on the clinical course of asthma in adults (table - ) , and as airway obstruction becomes fixed with advancing age, separating asthma from copd becomes increasingly difficult. in the study by schachter and coworkers, of the male participants age years or older with asthma, % improved and only % worsened during follow-up. among female participants, % improved and none worsened during follow-up. bronniman and burrows followed asthmatics, who were drawn from the general population sample in tucson, arizona, of persons, over a -year period. participants were classified as in remission if they had active disease at baseline and on follow-up denied medication use, asthma attacks, and frequent attacks of shortness of breath with wheezing during the preceding year. after years of follow-up, % were in remission, with the highest rate found among those between and years of age at enrollment ( %) and the lowest rate found for those between and years of age ( %). remission was more common in those with less frequent wheezing, less frequent asthma attacks, and less frequent attacks of shortness of breath with wheezing. remission was significantly less likely increased decline in persons with asthma compared with controls and others showing no difference between asthmatics and controls. peat and woolcock followed persons with asthma, who were to years old on enrollment, and control participants from busselton, australia. the asthmatic individuals had lower lung function values at enrollment and the fev declined at ml/yr more in the persons with asthma compared with the controls. schachter and colleagues followed the lung function of persons with asthma and with wheezing. over a -year interval, there was a similar excess loss of fev in the persons with asthma. ulrik and lange followed subjects over a -year period and found that asthmatic subjects had lower baseline lung function values and an excess annual decline in fev compared with nonasthmatics; the excess annual decline was ml in asthmatic men and ml in asthmatic women. some individuals with asthma appear to eventually develop irreversible airflow obstruction, which has been related to duration and severity of asthma. , a continuing effect of asthma was found when follow-up was extended to years. other studies have not shown increased loss of function associated with having a diagnosis of asthma. burrows and colleagues examined the course of asthma over years in asthmatics from the general population and compared them with two other groups: copd subjects and subjects who did not fit clearly into either group. the asthmatic subjects had a ml/yr decline in fev , compared with a -ml decline years. this was a highly selected group with many comorbidities, which probably influenced the eventual outcome. unfortunately, little is known about the outcome of elderly asthmatics that are not as ill. panhuysen and colleagues followed persons with asthma over years. the participants had been comprehensively evaluated in an asthma clinic in the netherlands between and at the ages of to years (mean, years). on retesting, % no longer showed bronchial hyperresponsiveness on histamine challenge, and based on a lack of bronchial hyperresponsiveness, symptoms, and lung function level, % were considered to no longer have asthma. settipane and colleagues followed a group of college students over years. half of those with asthma on follow-up reported the disease as inactive, although about % of the new cases occurred during follow-up. in those with chronic productive cough or a coexisting diagnosis of chronic bronchitis or emphysema. a normal level of percent predicted fev at baseline was the most powerful predictor of remission. persons to years old with active symptoms had only a % remission rate over years. broder and colleagues reported similar findings, with a remission rate of . % for patients between and years of age and % in those years of age or older. all relapse rates increased with age. braman and coworkers investigated the outcomes of nonsmoking asthmatics older than years of age. all had moderately severe asthma requiring daily bronchodilators, and of the needed daily glucocorticoids. over years, dependency on those drugs and complications of therapy were common. no patients died of acute asthma, but two died of chronic respiratory failure. mortality was not related to the level of pulmonary function or duration of asthma in philadelphia: wb saunders number of allergens to be tested to assess allergenic sensitization in epidemiologic studies: results of the european community respiratory health survey i total ige levels and asthma prevalence in the us population: results from the national health and nutrition examination survey recognition of the allergic phenotypenatural history of atopy measuring atopy in a multi-centre epidemiological study terminology, definitions, and classification of chronic pulmonary emphysema and related conditions: a report of the conclusions of a ciba guest symposium meaning of diagnostic terms in broncho-pulmonary disease chronic bronchitis, asthma, and pulmonary emphysema world health organization. epidemiology of chronic nonspecific respiratory diseases standardization of spirometry- update expert panel report on guidelines for diagnosis and management of asthma national heart, lung, and blood institute/ world health organization. global initiative for asthma public health service, national institutes of health (nih), et al. practical guide for the diagnosis and management of asthma. publication no. - national heart, lung, and blood institute. global initiative for asthma. global strategy for asthma management and prevention. publication no. - underdiagnosis and undertreatment of asthma in childhood the effects of death certification and coding practices on observed differences in respiratory disease mortality in e.e.c. countries the effect of death certification practice on recorded national asthma mortality rates international study of asthma and allergies in childhood (isaac): rationale and methods what symptoms predict the bronchial response to histamine? evaluation in a community survey of the bronchial symptoms questionnaire ( ) of the international union against tuberculosis and lung disease validity and repeatability of the iuatld ( ) bronchial symptoms questionnaire: an international comparison variations in the prevalence of respiratory symptoms, self-reported asthma attacks, and use of asthma medication in the european community respiratory health survey (ecrhs) national income, self-reported wheezing and asthma diagnosis from the world health survey comparison of a video questionnaire with the iuatld written questionnaire for measuring asthma prevalence comparison of bronchial reactivity and peak expiratory flow variability measurements for epidemiological studies methods for assessing bronchial reactivity bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance estimation and repeatability of the response to inhaled histamine in a community survey bronchial hyperreactivity descriptive epidemiology of bronchial reactivity in an adult population: results from a community study allergic rhinitis: definition, epidemiology, pathophysiology, detection, and diagnosis international primary care respiratory group guidelines: management of allergic rhinitis european position paper on rhinosinusitis and nasal polyps european position paper on rhinosinusitis and nasal polyps reliability of ep os symptom criteria and nasal endoscopy in the assessment of chronic rhinosinusitis-a ga len study the evaluation and management of food allergy in atopic dermatitis validation of the u.k. diagnostic criteria for atopic dermatitis in a population setting. u.k. diagnostic criteria for atopic dermatitis working party reported adverse food reactions overestimate true food allergy in the community the distribution of total and specific ige in the european community respiratory health survey international variation in prevalence of rhinitis and its relationship with sensitisation to perennial and seasonal allergens international variations in associations of allergic markers and diseases in children: isaac phase two allergy and parasites: the measurement of total and specific ige levels in urban and rural communities in rhodesia atopy, asthma, and antibodies to ascaris among rural and urban children in kenya effect of body mass on exercise-induced bronchospasm and atopy in african children global initiative for asthma (gina) program. the global burden of asthma: executive summary of the gina dissemination committee report worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: isaac national trends in the morbidity and mortality of asthma in the us. prevalence, hospitalization and death from asthma over two decades: - behavioral risk factor surveillance system survey: current asthma prevalence data. atlanta: us department of health and human services trends in asthma prevalence, health care use, and mortality in the united states development of seasonal allergic rhinitis during the first years of life natural history of hay fever: a -year follow-up of college students chronic rhinosinusitis in europe-an underestimated disease. a ga len study worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: isaac phases one and three repeat multicountry cross-sectional surveys isaac phase three study group. global variations in prevalence of eczema symptoms in children from isaac phase three the prevalence of food allergy: a meta-analysis food allergy among children in the united states the prevalence, severity, and distribution of childhood food allergy in the united states prevalence of challenge-proven ige-mediated food allergy using population-based sampling and predetermined challenge criteria in infants incidence of parentally reported and clinically diagnosed food hypersensitivity in the first year of life prevalence of sensitization reported and objectively assessed food hypersensitivity amongst sixyear-old children: a population-based study prevalence of sensitization to food allergens, reported adverse reaction to foods, food avoidance, and food hypersensitivity among teenagers prevalence and distribution of sensitization to foods in the european community respiratory health survey: a europrevall analysis prevalence of seafood allergy in the united states determined by a random telephone survey prevalence of peanut and tree nut allergy in the united states determined by means of a random digit dial telephone survey: a -year follow-up study us prevalence of self-reported peanut, tree nut, and sesame allergy: -year follow-up a population-based questionnaire survey on the prevalence of peanut, tree nut, and shellfish allergy in asian populations eosinophilic esophagitis eosinophilic esophagitis: escalating epidemiology? swiss eoe study group. escalating incidence of eosinophilic esophagitis: a -year prospective, population-based study in olten county, switzerland longitudinal changes in allergen skin test reactivity in a community population sample change in prevalence of ige sensitization and mean total ige with age and cohort changes in atopy over a quarter of a century, based on cross sectional data at three time periods prevalence and natural history of asthma in schoolchildren asthma and airway hyperresponsiveness among belgian conscripts, - total population survey of the frequency and severity of asthma in year old boys in an urban area in israel prevalence of asthma and allergy in schoolchildren in belmont, australia: three cross sectional surveys over years british thoracic society research committee. asthma prevalence in prevalence of asthma and bronchial hyperreactivity in danish schoolchildren: no change over years exercise-induced bronchospasm and atopy in ghana: two surveys ten years apart increasing prevalence of bronchial hyperresponsiveness in three selected areas in east germany. bitterfeld study group summary health statistics for u.s. children: national health interview survey us childhood asthma prevalence estimates: the impact of the national health interview survey redesign national health interview survey (nhis). asthma questions respiratory diseases in minorities of the united states surveillance for asthma-united states asthma prevalence, health care use, and mortality: united states early-life risk factors and incidence of rhinitis: results from the european community respiratory health study-an international population-based cohort study vital and health statistics, series increasing prevalence of hay fever and atopy among children in leipzig, east germany increase of asthma and allergic rhinitis prevalence in young italian men increasing prevalence of allergic rhinitis symptoms in an adult danish population intergenerational year trends in the prevalence of asthma and hay fever in adults: the midspan family study surveys of parents and offspring changing trends in asthma in - year olds between comparison of food allergy prevalence among chinese infants in chongqing increasing hospital admissions for systemic allergic disorders in england: analysis of national admissions data time trends in allergic disorders in the uk food allergy among u.s. children: trends in prevalence and hospitalizations rising prevalence of allergy to peanut in children: data from sequential cohorts genetic factors in nonspecific bronchial hyperreactivity segregation analysis of physician diagnosed asthma in hispanic and non-hispanic white families segregation analysis of physician-diagnosed asthma in hispanic and non-hispanic white females. a recessive component? adult patients may outgrow their asthma: a -year follow-up study familial occurrence of chronic respiratory disease and familial resemblance in ventilatory capacity family aggregation of pulmonary function measurements influence of personal and family factors on ventilatory function of children social and familial factors in the development of early childhood asthma factors influencing the prevalence of asthma among first degree relatives of extrinsic and intrinsic asthmatics genetic analysis of allergic disease in twins allergy in twin pairs a population-based study of bronchial asthma in adult twin pairs a large-scale, consortium-based genomewide association study of asthma risk factors for asthma incidence. a review of recent prospective evidence sex differences in factors associated with childhood-and adolescent-onset wheeze maximal expiratory flows at functional residual capacity: a test of lung function for young children specific airway resistance from the perinatal period into adulthood. alterations in childhood pulmonary disease epidemiology of acute lower respiratory disease in children acute respiratory illness in an american community. the tecumseh study diminished lung function as a predisposing factor for wheezing respiratory illness in infants gender differences in pulmonary disease maternal folate levels in pregnancy and asthma in children at age years international variations in bronchial responsiveness in children: findings from isaac phase two respiratory disorders and allergy skin-test reactions association of asthma with serum ige levels and skin-test reactivity to allergens risk factors for childhood respiratory disease: the effect of host factors and home environmental exposures atopy and wheeze in children according to parental atopy and family size parental asthma, parental eczema, and asthma and eczema in early childhood relation between airway responsiveness and serum ige in children with asthma and in apparently normal children relation between airway responsiveness and serum ige in children with asthma and in apparently normal children the natural history of asthma a diet rich in sodium may potentiate asthma. epidemiologic evidence for a new hypothesis higher serum folate levels are associated with a lower risk of atopy and wheeze nutritional epidemiology diet as a risk factor for asthma confounding and effect modification in studies of diet and childhood asthma and allergies those confounded vitamins: what can we learn from the differences between observational versus randomized trial evidence? nutrients and foods for the primary prevention of asthma and allergy: systematic review and meta-analysis antioxidants and allergic disease: a case of too much or too little? diet and asthma: nutritional implications from prevention to treatment prenatal and childhood mediterranean diet and the development of asthma and allergies in children fatty acid composition abnormalities in atopic disease: evidence explored and role in the disease process examined omega and oils for primary prevention of allergic disease: systematic review and meta-analysis vitamin d and asthma: scientific promise and clinical reality asthma mortality in england and wales: evidence for a further increase, - effect of changing dietary sodium on the airway response to histamine effect of dietary salt on bronchial reactivity to histamine in asthma respiratory symptoms and bronchial responsiveness are related to dietary salt intake and urinary potassium excretion in male children dietary sodium intake and the risk of airway hyperreactivity in a random adult population methacholine airway responsiveness and -hour urine excretion of sodium and potassium. the normative aging study dietary magnesium, lung function, wheezing, and airway hyperreactivity in a random adult population sample dietary sodium manipulation and asthma diet as a risk factor for atopy and asthma diet and obstructive lung diseases the role of diet in the aetiology of asthma fish oil supplementation in pregnancy and lactation may decrease the risk of infant allergy effect of n- long chain polyunsaturated fatty acid supplementation in pregnancy on infants' allergies in first year of life: randomised controlled trial the vitamin d slant on allergy is vitamin d deficiency to blame for the asthma epidemic? a case control study of vitamin d status and asthma in adults dairy food, calcium and vitamin d intake and prevalence of allergic disorders in pregnant japanese women relationship between serum vitamin d, disease severity, and airway remodeling in children with asthma vitamin d deficiency as a strong predictor of asthma in children decreased serum ll- and vitamin d levels in atopic dermatitis: relationship between il- and oncostatin m vitamin d serum levels in allergic rhinitis: any difference from normal population? vitamin d levels and food and environmental allergies in the united states: results from the national health and nutrition examination survey serum -hydroxy-vitamin d and ionised calcium in relation to lung function and allergen skin tests age-and atopy-dependent effects of vitamin d on wheeze and asthma serum vitamin d levels and markers of severity of childhood asthma in costa rica vitamin d levels, lung function, and steroid response in adult asthma correlation between serum -hydroxyvitamin d levels and severity of atopic dermatitis in children princess anne hospital study group. maternal vitamin d status during pregnancy and childhood outcomes does vitamin d intake during infancy promote the development of atopic allergy? infant vitamin d supplementation and allergic conditions in adulthood: northern finland birth cohort serum micronutrient concentrations and childhood asthma: the piama birth cohort study vitamin d and atopy and asthma phenotypes in children: a longitudinal cohort study primary prevention of asthma and allergy the role of breastfeeding in the development of allergies and asthma effects of early nutritional interventions on the development of atopic disease in infants and children: the role of maternal dietary restriction, breastfeeding, timing of introduction of complementary foods, and hydrolysed formulas the australasian society of clinical immunology and allergy position statement: summary of allergy prevention in children exclusive breastfeeding and incident atopic dermatitis in childhood: a systematic review and meta-analysis of prospective cohort studies systematic review and meta-analysis investigating breast feeding and childhood wheezing illness prevalence of overweight and obesity in the united states complex interactions in complex traits: obesity and asthma obesity: insight into the origins of asthma obesity and asthma prospective study of body mass index, weight change, and risk of adult-onset asthma in women relation of body mass index to asthma and atopy in children: the national health and nutrition examination study iii relation of two different subtypes of croup before age three to wheezing, atopy, and pulmonary function during childhood: a prospective study overweight, obesity, and incident asthma a meta-analysis of prospective epidemiologic studies childhood body mass index and risk of asthma in adolescence: a systematic review overdiagnosis of asthma in obese and nonobese adults body mass indexpercentile and diagnostic accuracy of childhood asthma asthma and atopy in overweight children association of obesity with ige levels and allergy symptoms in children and adolescents: results from the national health and nutrition examination survey obesity and the prevalence of allergic diseases in schoolchildren association between atopic dermatitis and obesity in adulthood comparability of parent-reported respiratory illnesses to clinical diagnoses lung function abnormalities after acute bronchiolitis pulmonary function abnormalities in symptom-free children after bronchiolitis study of -year-old children with a history of respiratory syncytial virus bronchiolitis in infancy increased incidence of bronchial reactivity in children with a history of bronchiolitis wheezing, asthma, and pulmonary dysfunction years after infection with respiratory syncytial virus in infancy respiratory problems years after acute bronchiolitis in infancy long-term prospective study in children after respiratory syncytial virus infection lower respiratory tract illness in the first two years of life: epidemiologic patterns and costs in a suburban pediatric practice severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age hospitalization for rsv bronchiolitis before months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study the relationship of respiratory infections in early childhood to the occurrence of increased levels of bronchial responsiveness and atopy predictors of repeated wheeze in the first year of life: the relative roles of cockroach, birth weight, acute lower respiratory illness, and maternal smoking respiratory syncytial virus in early life and risk of wheeze and allergy by age years exploring the association between severe respiratory syncytial virus infection and asthma: a registry-based twin study rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in high-risk children decreased lung function after preschool wheezing rhinovirus illnesses in children at risk to develop asthma asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient type iii interferon-l production in asthma exacerbations the influence of neonatal lung function on rhinovirus-associated wheeze sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution a novel group of rhinoviruses is associated with asthma hospitalizations association between human rhinovirus c and severity of acute asthma in children clinical spectrum of human rhinovirus infections in hospitalized hong kong children high titers of ige antibody to dust mite allergen and risk for wheezing among asthmatic children infected with rhinovirus childhood asthma after bacterial colonization of the airway in neonates of attraction and rejectionasthma and the microbial world does antibiotic exposure during infancy lead to development of asthma? a systematic review and metaanalysis infection: friend or foe in the development of atopy and asthma? the epidemiological evidence the intestinal microflora in allergic estonian and swedish -year-old children antibiotic use during infancy promotes a shift in the t(h) /t(h) balance toward t(h) -dominant immunity in mice antibiotic use during the first year of life and asthma relation of early antibiotic use to childhood asthma: confounding by indication? prenatal or early-life exposure to antibiotics and risk of childhood asthma: a systematic review infant antibiotic use and wheeze and asthma risk: a systematic review and meta-analysis pulmonary disease following respirator therapy of hyaline-membrane disease. bronchopulmonary dysplasia late pulmonary sequelae of bronchopulmonary dysplasia the long-term pulmonary sequelae of prematurity: the role of familial airway hyperreactivity and the respiratory distress syndrome associations between family history of asthma, bronchopulmonary dysplasia, and childhood asthma in very low birth weight children respiratory symptoms at age years in a cohort of very low birth weight children determinants of bronchial responsiveness at school age in prematurely born children very low birthweight and asthma by age seven years in a national cohort birth weight < g and respiratory health at age impact of low birth weight on early childhood asthma in the united states prematurity as a risk factor for asthma in preadolescent children pulmonary function in children with initial low birth weight birth characteristics and asthma symptoms in young adults: results from a population-based cohort study in norway perinatal risk factors for asthma in finnish adolescent twins low birth weight and preterm delivery as risk factors for asthma and atopic dermatitis in young adult males pre-and perinatal risk factors for asthma in inner city african-american children longitudinal follow-up of lung function from childhood to adolescence in prematurely born patients with neonatal chronic lung disease is there a link between wheezing in early childhood and adverse birth outcomes? a systematic review preterm delivery and asthma: a systematic review and meta-analysis definition and classification of asthma aetiological agents in occupational asthma diisocyanate asthma and gene-environment interactions with il ra, cd- , and il- genes occupational asthma diagnosis and management of work-related asthma: american college of chest physicians consensus statement standards of care for occupational asthma occupation and asthma: a population-based incident casecontrol study occupational exposures and asthma in , adults from the general population occupational exposures associated with workrelated asthma and work-related wheezing among u.s. workers occupational asthma in europe and other industrialised areas: a population-based study. european community respiratory health survey study group incidence of work-related asthma in members of a health maintenance organization how much adult asthma can be attributed to occupational factors? clean air act . wasington, d.c.: environmental protection agency respiratory health effects of air pollution: update on biomass smoke and traffic pollution the role of air pollution in asthma and other pediatric morbidities asthma and air quality acute effects of motor vehicle traffic-related air pollution exposures on measures of oxidative stress in hman airways air pollution and airway disease traffic-related air pollution and asthma onset in children: a prospective cohort study with individual exposure measurement trafficrelated air pollution and the development of asthma and allergies during the first years of life childhood incident asthma and traffic-related air pollution at home and school the effect of air pollution on lung development from to years of age community outbreaks of asthma associated with inhalation of soybean dust. toxicoepidemiological committee casecontrol study of serum immunoglobulin-e antibodies reactive with soybean in epidemic asthma preventing asthma epidemics due to soybeans by dustcontrol measures reexamination of epidemic asthma in new orleans, louisiana, in relation to the presence of soy at the harbor air pollution and health indoor air quality handbook epidemiological studies of ozone exposure effects epidemiology of particle effects air quality criteria for particulate matter. epa/ /p- / ad and bd air quality criteria for ozone and related photochemical oxidants (final) effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects effect of nitrogen dioxide and sulfur dioxide on airway responses of mild asthmatic patents to allergen inhalation research priorities for airborne particulate matter: no. . immediate priorities and a long-range research portfolio association of low-level ozone and fine particles with respiratory symptoms in children with asthma epidemiology of pollution-induced airway disease: urban/rural differences in east and west germany prevalence of respiratory and atopic disorders among children in the east and west of germany five years after unification prevalence of asthma and atopy in two areas of west and east germany adverse effects of low-level air pollution on the respiratory health of schoolchildren in hong kong inner city air pollution and respiratory health and atopy in children association between indoor and outdoor air pollution and adolescent asthma from to in taiwan traffic-related air pollutants, and asthma prevalence in middle-school children in taiwan prevalence of asthma and mean levels of air pollution: results from the french paarc survey. pollution atomospherique et affections respiratoires chroniques respiratory health and individual estimated exposure to traffic-related air pollutants in a cohort of young children traffic, susceptibility, and childhood asthma estimated long-term ambient concentrations of pm and development of respiratory symptoms in a nonsmoking population long-term particulate and other air pollutants and lung function in nonsmokers is ambient air pollution associated with the incidence of asthma? systematic review and metaanalysis of epidemiological evidence clearing the air: asthma and indoor air exposures a longitudinal study of indoor nitrogen dioxide levels and respiratory symptoms in inner-city children with asthma indoor particulate matter increases asthma morbidity in children with non-atopic and atopic asthma the indoor environment and its effects on childhood asthma indoor environment and children's health: recent developments in chemical, biological, physical and social aspects indoor air pollution. a health perspective a review of the epidemiological evidence on health effects of nitrogen dioxide exposure from gas stoves domestic exposure to formaldehyde significantly increases the risk of asthma in young children association of domestic exposure to volatile organic compounds with asthma in young children association between personal exposure to volatile organic compounds and asthma among us adult population quantitative assessments of indoor air pollution and respiratory health in a population-based sample of french dwellings residential exposure to volatile organic compounds and asthma the influence of maternal exposure to volatile organic compounds on the cytokine secretion profile of neonatal t cells committee on the health effects of indoor allergens, division of health promotion and disease prevention. indoor allergens: assessing the controlling adverse health effects exposure to house-dust mite allergen (der p i) and the development of asthma in childhood. a prospective study perennial allergen sensitisation early in life and chronic asthma in children: a birth cohort study exposure to dogs and cats in the first year of life and risk of allergic sensitization at to years of age early allergen exposure, skin prick responses, and atopic wheeze at age in english children: a cohort study early exposure to house-dust mite and cat allergens and development of childhood asthma: a cohort study. multicentre allergy study group home dampness and molds, parental atopy, and asthma in childhood: a six-year populationbased cohort study indoor molds and asthma in adults the medical effects of mold exposure differential effects of outdoor versus indoor fungal spores on asthma morbidity in inner-city children housing interventions and control of asthma-related indoor biologic agents: a review of the evidence the canadian childhood asthma primary prevention study: outcomes at years of age primary prevention of asthma and atopy during childhood by allergen avoidance in infancy: a randomised controlled study prevention of asthma during the first years of life: a randomized controlled trial is there any role for allergen avoidance in the primary prevention of childhood asthma us department of health and human services (usdhhs). the health effects of active smoking: a report of the surgeon general us department of health and human services (usdhhs). the health benefits of smoking cessation: a report of the surgeon general government printing office asthma and cigarette smoking: a review of the empirical literature us department of health and human services (usdhhs). the health effects of involuntary exposure to tobacco smoke environmental toxicants: human exposures and their health effects. new york: van nostrand reinhold preliminary data: exposure of persons aged ≥ years to tobacco smoke-united states national report on human exposure to environmental chemicals.results. nhanes iv. cas no. - - us department of health and human services (usdhhs). the health consequences of involuntary smoking: a report of the surgeon general government printing office the influence of a family history of asthma and parental smoking on airway responsiveness in early infancy the effect of maternal smoking during pregnancy on early infant lung function antenatal determinants of neonatal immune responses to allergens maternal smoking in pregnancy alters neonatal cytokine responses prenatal and passive smoke exposure and incidence of asthma and wheeze: systematic review and meta-analysis maternal and grandmaternal smoking patterns are associated with early childhood asthma allergic rhinitis: definition, epidemiology, pathophysiology, detection, and diagnosis epidemiology of physician-diagnosed allergic rhinitis in childhood gestational age at birth and risk of allergic rhinitis in young adulthood preterm birth reduces the incidence of atopy in adulthood birth by cesarean section, allergic rhinitis, and allergic sensitization among children with a parental history of atopy reduced diversity of the intestinal microbiota during infancy is associated with increased risk of allergic disease at school age a genome-wide meta-analysis of genetic variants associated with allergic rhinitis and grass sensitization and their interaction with birth order acetaminophen use and risk of asthma, rhinoconjunctivitis, and eczema in adolescents: international study of asthma and allergies in childhood phase three oral contraception, body mass index, and asthma: a cross-sectional nordic-baltic population survey atopic diseases, allergic sensitization, and exposure to traffic-related air pollution in children perinatal predictors of atopic dermatitis occurring in the first six months of life filaggrin mutations associated with skin and allergic diseases support for variants of eczema endotoxin exposure and eczema in the first year of life endotoxin in inner-city homes: associations with wheeze and eczema in early childhood increased risk of eczema but reduced risk of early wheezy disorder from exclusive breastfeeding in high-risk infants impact of early feeding on childhood eczema: development after nutritional intervention compared with the natural course-the giniplus study up to the age of years the introduction of allergenic foods and the development of reported wheezing and eczema in childhood: the generation r study lack of evidence for a protective effect of prolonged breastfeeding on childhood eczema: lessons from the international study of asthma and allergies in childhood (isaac) phase two solid food introduction in relation to eczema: results from a four-year prospective birth cohort study global analysis of breast feeding and risk of symptoms of asthma, rhinoconjunctivitis and eczema in - year old children: isaac phase three probiotics in pregnant women to prevent allergic disease: a randomized, double-blind trial a differential effect of probiotics in the prevention of eczema and atopy: a double-blind, randomized, placebo-controlled trial the evaluation and management of food allergy in atopic dermatitis is delivery by cesarean section a risk factor for food allergy? cohort study of peanut and tree nut sensitisation by age of years natural history and course of asthma . reed ce. the natural history of asthma in adults: the problem of irreversibility airway remodeling contributes to the progressive loss of lung function in asthma: an overview the natural history of asthma tucson children's respiratory study: to present a birth cohort study of subjects at risk of atopy: twenty-two-year follow-up of wheeze and atopic status spectrum of asthma in children. i. clinical and physiological components incidence and prognosis of asthma and wheezing illness from early childhood to age in a national british cohort prevalence, natural history, and relationship of wheezy bronchitis and asthma in children. an epidemiological study the melbourne asthma study: - childhood asthma in adult life: a further study at years of age asthma from birth to age : incidence and relation to prior and concurrent atopic disease ventilatory function in british adults after asthma or wheezing illness at ages - follow-up of asthma from childhood to adulthood: influence of potential childhood risk factors on the outcome of pulmonary function and bronchial responsiveness in adulthood a longitudinal, population-based, cohort study of childhood asthma followed to adulthood rate of decline of lung function in subjects with asthma a prospective study of asthma in a rural community decline of lung function in adults with bronchial asthma the relationship between age and duration of asthma and the presence of persistent obstruction in asthma interacting effects of atopy and bronchial hyperresponsiveness on the annual decline in lung function and the exacerbation rate in asthma a -year follow-up study on ventilatory function in adults with asthma the course and prognosis of different forms of chronic airways obstruction in a sample from the general population a prospective study of the natural history of asthma. remission and relapse rates epidemiology of asthma and allergic rhinitis in a total community asthma in the elderly. a comparison between patients with recently acquired and long-standing disease natural history of asthma: a -year followup of college students incidence and remission of asthma in schoolchildren: report from the obstructive lung disease in northern sweden studies a prospective study of asthma incidence and its predictors: the rhine study onset and remission of allergic rhinitis and asthma and the relationship with atopic sensitization and smoking incidence of asthma and its determinants among adults in spain incidence of asthma and mortality in a cohort of young adults: a -year prospective study environmental tobacco smoke exposure and nocturnal symptoms among inner-city children with asthma incidence of asthma and respiratory symptoms by sex, age and smoking in a community study early life risk factors for current wheeze, asthma, and bronchial hyperresponsiveness at years of age a -year follow up of a birth cohort study prognosis of asthma in childhood incidence and remission of asthma: a retrospective study on the natural history of asthma in italy comparison of a beta -agonist, terbutaline, with an inhaled steroid, budesonide, in newly detected asthma tenor study group. gender differences in igemediated allergic asthma in the epidemiology and natural history of asthma: outcomes and treatment regimens (tenor) study a comparison of bronchodilator therapy with or without inhaled corticosteroid therapy in obstructive airways natural history of asthma in childhood-a birth cohort study physician-diagnosed asthma and allergic rhinitis in manitoba: - food allergy as a risk factor for lifethreatening asthma in childhood: a casecontrolled study outcome of wheeze in childhood. symptoms and pulmonary function years later a sevenyear follow-up study of adults with bronchial asthma respiratory symptoms in young adults should not be overlooked lung function in young adults who had asthma in childhood childhood asthma and lung function in mid-adult life association between allergy and asthma from childhood to middle adulthood in an australian cohort study preschool wheezing and prognosis at wheezy bronchitis in childhood: a distinct clinical entity with lifelong significance? the state of childhood asthma in young adulthood decline in lung function in the busselton health study: the effects of asthma and cigarette smoking the characteristics of bronchial asthma among a young adult population mortality and decline in lung function in adults with bronchial asthma: a ten year follow up key: cord- - abypk o authors: asner, sandra a.; petrich, astrid; hamid, jemila s.; mertz, dominik; richardson, susan e.; smieja, marek title: clinical severity of rhinovirus/enterovirus compared to other respiratory viruses in children date: - - journal: influenza other respir viruses doi: . /irv. sha: doc_id: cord_uid: abypk o background: human rhinovirus/enterovirus (hrv/ent) infections are commonly identified in children with acute respiratory infections (aris), but data on their clinical severity remain limited. objectives: we compared the clinical severity of hrv/ent to respiratory syncytial virus (rsv), influenza a/b (flu), and other common respiratory viruses in children. patients/methods: retrospective study of children with aris and confirmed single positive viral infections on mid‐turbinate swabs by molecular assays. outcome measures included hospital admission and, for inpatients, a composite endpoint consisting of intensive care admission, hospitalization > days, oxygen requirements or death. results: a total of hrv/ent, rsv, flu, and other common respiratory viruses were identified. children with single hrv/ent infections presented with significantly higher rates of underlying immunosuppressive conditions compared to those with rsv ( · % versus · %; p < · ), flu ( · % versus %; p = · ) or any other single viral infection ( · % versus · %; p = · ). in multivariable analysis adjusted for underlying conditions and age, children with hrv/ent infections had increased odds of hospitalization compared to children with rsv infections (or · ; % ci · , · ; p < · ) or flu infections (or · ; % ci · , · ; < · ) and increased odds of severe clinical disease among inpatients (or · ; % ci · , · ; p = · ) when compared to those with flu infections. conclusions: children with hrv/ent had a more severe clinical course than those with rsv and flua/b infections and often had significant comorbidities. these findings emphasize the importance of considering hrv/ent infection in children presenting with severe acute respiratory tract infections. human rhinovirus/enterovirus (hrv/ent) has been recently identified as the leading pathogen in acute asthma exacerbations, bronchiolitis, and viral pneumonia, although the clinical severity of respiratory illnesses attributed to hrv/ent remains uncertain. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] studies conducted among younger children admitted for bronchiolitis reported conflicting information about the clinical severity of hrv/ent infections, possibly as a result of different age groups studied. , a study by papadopulos et al., reported that hrv/ent was a significant predictor for severe disease, another study by midulla et al. reported that hrv/ent bronchiolitis resulted in less severe disease compared to rsv bronchiolitis among infants. conversely, studies involving older hospitalized children , , consistently reported equivalent disease severity between respiratory illnesses caused by hrv/ent and those caused by other common respiratory viruses. explanations for the discrepancies observed among these studies , , include the use of different clinical criteria to assess clinical severity (clinical severity scores versus clinical outcomes) and the focus on different patient populations and age groups (inpatients and young children). furthermore, the contribution of hrv/ent infection versus host-related factors such as underlying immunosuppression to the severity of the disease was not reported in most studies. in summary, there is growing evidence that hrv/ent is the most commonly identified virus in lrtis in both adults and children and thus needs to be regarded as a pathogen responsible for much more than just the common cold. the objective of this study was to compare the clinical characteristics and the severity of illness of hrv-/ent-positive children to those positive for rsv a/b, flu-a/ flu-b, and other respiratory viruses by (i) using a large dataset with a broad pediatric inpatient and outpatient population; (ii) considering several clinical endpoints to assess disease severity, and (iii) conducting multivariable analysis to adjust for potential confounding variables. we conducted a single-center retrospective study of children presenting with an acute respiratory illness and any single viral infection documented by molecular assays from midturbinate swabs to the hospital for sick children, toronto, canada. multiplex pcr was conducted in a random sample of all patients presenting with arti in whom the treating physician ordered a mid-turbinate swab. testing for respiratory viruses relied on clinical assessment as routinely done in children with respiratory illnesses. these included common cold, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, and community-acquired pneumonia (cap). we used a dataset from the microbiology laboratory at the hospital for sick children, which originally aimed to compare the sensitivities of four different multiplex assays: resplex ii v . (qiagen, mississauga, on, canada); seeplex rv kit (seegene inc., seoul, korea); xtag-rvp and xtag-rvp fast, (luminex, austin, tx, usa) in addition to dfa and viral culture. the results from the original study were recently published elsewhere. the dataset was augmented through retrospective chart review. children with documented viral coinfections (i.e., detection of more than one viral pathogen in the same sample) were excluded as their inclusion would not allow single viral infection comparisons as a result of not being able to determine which of the viruses detected was the leading pathogen. data were collected from november to april and january to march , the times when multiplex pcr testing was routinely utilized in randomly selected patients under years of age presenting with acute respiratory tract infections (artis). urti was defined as the detection of any single respiratory viral infection together with symptoms involving the upper respiratory tract (nose and pharynx). lrti was defined as any patient with cough, tachypnea, and/ or any respiratory distress or wheezing. communityacquired pneumonia (cap) was defined as any of the above lrti symptoms with pulmonary infiltrates diagnosed on a chest x-ray by a radiologist. severe clinical disease was defined by hospital admission for outpatients and by a composite endpoint including intensive care (icu) admission, hospitalization > days, oxygen requirements or death in patients admitted to the hospital. ethics approval was obtained from the research ethics board at the hospital for sick children. information on patient demographics, relevant baseline characteristics such as underlying comorbidities and outcomes were all extracted from health records. relevant underlying comorbidities were grouped into three mutually exclusive categories: cardiorespiratory, prematurity, and any immunosuppressive/metabolic conditions. one or more of the following immunodeficiency states were included in the latter group: hematopoietic stem cell transplant (allogeneic and autologous) and solid organ transplant (sot) recipients, recipients of cancer chemotherapy or long-term immunosuppression for any chronic disease, and congenital immunodeficiency states. metabolic conditions included any inherited metabolic diseases such as cystinuria, phenylketonuria (pku), gout, and thyroid disease. in case of multiple comorbidities, patients were referred to the group considered as the most significant comorbidity: an underlying immunocompromised/metabolic condition was considered most significant comorbidity, followed by a cardiorespiratory comorbidity. outcomes consisted of hospital admission, duration of hospitalization, admission length > days, intensive care (icu) admission, any supplemental oxygen requirements, presence of urti, cap, and all-cause mortality. from november to april and january to march , mid-turbinate swabs (floqswabs, copan italia, brescia, italy) were tested for respiratory viruses of which ( %) were randomly selected each week among all specimens collected from children with acute respiratory tract infections (artis)) and submitted to the clinical laboratory for routine virology testing including molecular assays. neither laboratory staff nor clinicians were aware in advance which patient would be selected for viral detection by molecular assays. swabs were assayed by four different nucleic acid amplification-based assays: resplex ii v . , a true positive result was defined as two or more positive test results from among the four molecular assays. bacterial coinfection was defined as the presence of any bacterial pathogen, identified by culture from blood cultures or respiratory samples (bronchoalveolar lavage, tracheal secretions, sputum samples or pleural fluid) upon initial consultation with respiratory symptoms or within days of their initial consultation. staphylococcus epidermidis positive blood cultures were considered as pathogens if drawn from more than one peripheral blood culture, from one blood culture drawn from a central line, or from one peripheral line in high-risk patients such as those with underlying immunosuppressive conditions, prosthetic devices, or newborns according to recent guidelines. in addition, a positive urinary antigen for streptococcus pneumonia retrieved upon admission or within days of admission was also considered as bacterial coinfection. positive bacterial urinary or stool cultures and bacterial pathogen identified from skin or wound swabs were not considered as bacterial coinfections. standard descriptive and comparative statistics were performed on data categorized by viral pathogen, where hrv/ ent was used as the reference and compared to rsv, flu, or category consisting of all other common viruses including piv - , hmpv, hbov, adv, and coronaviruses. the chisquare test or fisher's exact test was used to compare categorical variables between groups as appropriate. multivariable logistic regression was used to compare the clinical correlates of clinical disease severity between children infected with hrv/ent and those infected with either rsv, flu, or other common single respiratory viruses. we derived medians, used the mann-whitney nonparametric method for comparisons of non-normally distributed continuous data, and used a transformation using the natural logarithm (ln) of the outcome variable when performing multivariable linear regression. a two-sided test was performed, and -a p value < Á was considered to be statistically significant. data were analyzed using spss statistical software (version . , spss inc, chicago, il, usa). a total of children evaluated for any respiratory illness were screened for respiratory viruses from mid-turbinate swabs by molecular assays during the study period. of these, ( Á %) children were detected positive for any respiratory virus. among these patients, ( Á %) were admitted, and among these inpatients, ( Á %) presented with severe disease. a total of ( Á %) had two or more respiratory viruses and were excluded from this analyses. the remaining, ( Á %), tested positive for a single respiratory virus: ( Á %) hrv/ent, ( Á %) rsv, ( Á %) flu, and ( Á %) other respiratory viruses. our rate of overall bacterial coinfection was low ( Á %). among inpatients, children with single hrv/ent infections were significantly older compared to those with single rsv infections (median age Á years versus Á years, p < Á ). children with hrv/ent infections presented with a significantly higher rate of underlying cardiorespiratory comorbidities compared to children with rsv ( Á % versus Á %; or Á ; % ci Á , Á ; p = Á ), flu ( Á % versus Á ; or ; % ci Á , Á ; p = Á ), or any other single viral infection ( Á % versus Á %; or Á ; % ci Á , Á ; p = Á ) ( table ) . children with hrv/ent infections had significantly higher rates of pneumonia compared to those with rsv ( Á % versus Á %; or Á ; % ci Á - Á ; p = Á ). when compared to children with rsv infections or flu infections, those with hrv/ent infections had significantly higher admission rates ( Á % versus, respectively, Á % and Á %; both p < Á ). among children who were admitted to hospital, those with hrv/ent infections had an increased length of stay compared to those with flu infections ( days, iqr - Á versus days; iqr - days; p = Á ). of six children who died, four presented with hrv/ent infections; one presented with a progressive acute respiratory distress syndrome (ards) after adv infection and died weeks after flu-a infection; and the remaining one presented with an hbov infection. of the four fatalities with hrv/ent infections, three presented with underlying immunosuppressive conditions and one with a cardiac comorbidity. two of the patients died from a sepsis-like picture with respiratory failure and possible underlying pneumonia with no other pathogens identified, thus suggesting that hrv/ent may have potentially contributed to the mortality. the remaining two patients likely died of their underlying disease ( table ) . in multivariable analysis adjusted for age and comorbidities, we identified that children with hrv/ent infections were significantly more likely to be hospitalized compared to those with rsv infections (or Á ; % ci Á , Á ; p < Á ) or flu infections (or Á ; % ci Á - Á ; < Á ) (tables ). furthermore, age (or Á per year; % ci Á , Á ; p = Á ) and underlying immunosuppressive/metabolic conditions (or Á ; % ci Á , Á ; p < Á ) were significant predictors of hospital admission. an interaction term measuring the combination of hrv-/ent-positive status and underlying immunocompromised condition was not significant, and its inclusion did not affect the risk estimates above. post hoc analyzes conducted among patients with an underlying immunosuppressive condition did not affect the risk estimates above although our results were not human rhinovirus-/enterovirus-infected children were significantly more likely to present with severe clinical disease in multivariable analysis compared with children admitted to hospital with flu infections (or Á ; % ci Á , Á ; p = Á ) (tables ). children admitted with hrv/ent infections had an % increase in the length of stay compared to those admitted with flu infections (b coefficient Á , % ci Á , Á ; p = Á ) in multivariable analysis, where the logarithm of admission length was used as an outcome variable. children with underlying immunosuppressive or metabolic states had a % increase in the length of admission compared to those without underlying immunosuppressive or metabolic conditions (b coefficient Á ; % ci Á , Á ; p < Á ) ( table ). three important observations made in our study were that hrv/ent acute respiratory infections were very commonly detected in our patient population; children with hrv/ent infections had more severe outcomes compared to those with other common respiratory viruses; and children with underlying cardiorespiratory or immunocompromised/metabolic conditions were more likely to present with hrv/ent infections. the proportion of single hrv/ent infections reported in our study ( Á %) was significantly higher than in studies which used conventional diagnostic methods ( %), , - but comparable to those in which molecular tests were used, including studies of children with artis ( Á %) , and hrv/ent, enterovirus/rhinovirus; rsv, respiratory syncytial virus; flu, influenza; others: piv, parainfluenza; hmpv, human meta-pneumovirus; adv, adenovirus, coronaviruses, hbov. values indicated in bold were considered as significant p < . . hrv/ent, enterovirus/rhinovirus; rsv, respiratory syncytial virus; flu, influenza; others: piv, parainfluenza; hmpv, human meta-pneumovirus; adv, adenovirus, coronaviruses, hbov. values indicated in bold were considered as significant p < . . infants with bronchiolitis ( %). these discrepancies can be attributed to the higher sensitivity and broader range of virus detection by molecular-based methods. similar to others studies, , , , children infected by hrv/ ent alone were younger compared to those with flu infections but older compared to those with rsv infections. in our study, children with hrv/ent infections presented with higher rates of underlying cardiorespiratory and immunosuppressive conditions compared to those with any other single viral infection. our findings are similar to those described in adults admitted with hrv/ent respiratory infections, in whom high rates of underlying immunosuppressive conditions were identified, thus suggesting that these patients may be at higher risk of hrv/ent infections. , [ ] [ ] [ ] the extent to which severity of respiratory illness is attributed to hrv/ent or to other underlying conditions is yet unclear. notwithstanding the high proportion of underlying comorbidities identified among our hrv/ent population, hrv-/ ent-positive status was identified as a significant independent predictor for hospital admission and resulted in an increased clinical disease severity among inpatients compared with either rsv or flu, while controlling for underlying comorbidities and age. furthermore, we found significantly longer duration of hospitalization among children admitted with hrv/ent infections compared to those admitted with flu infections. also, higher rates of pneumonia were observed among children with single hrv/ent infections compared to those with rsv single viral infections as suggested by a previous study by malcolm et al. finally, half of the deaths observed among the four hrv-/ent-infected children with underlying conditions may have resulted from their hrv/ ent respiratory illnesses as no other viral, bacterial, or fungal pathogens were documented. all these findings contrast with recently published studies , , which reported equivalent disease severity between subjects with hrv/ent infections and those with other common viral infections. these studies, however, had major limitations, which may have resulted in not detecting a true difference. firstly, the studies were conducted in inpatients only. one can speculate that hospitalized patients share a level of comorbidity leading to more similar outcomes regardless of the virus involved. furthermore, most of these studies compared hrv/ent infections to all other viruses combined and did not adjust for underlying comorbidities. while treatment of flu-a-positive children with oseltamivir might have been expected to improve their prognosis, no children included in this study received oseltamivir, as specimens were collected before the implementation of guidelines advocating for oseltamivir treatment of high-risk children thus, the difference in outcomes between hrv/ent and treated flu-a may be even more pronounced than found in our study. an important strength of our study was the inclusion of outpatients, thus enabling the use of hospital admission as a measure of clinical severity. furthermore, the adjustment for underlying comorbidities in multivariable analysis reinforced the association between hrv/ent and clinical outcomes. given our low rate of bacterial coinfections, no adjustment for this variable was made in multivariable analyses. finally, we had adequate sample size to compare hrv/ent to a number of different viruses. potential limitations of our study relate to its retrospective design. however, we believe that most of our patient-related important outcomes could be well assessed though chart review. similarly, the observational nature of our study may have led to selection bias as not all consecutive patients were tested for respiratory viruses as a result of limited resources. random sampling ensured an unbiased selection of samples tested by molecular assays. furthermore, the criteria for using virologic diagnostics as part of routine patient care may have varied during the study. for instance, viral culture was only routinely used from november to april , which may have affected the comparisons of illness severity between different viruses. third, we assessed the presence or absence of viruses, but did not measure their viral load; higher viral loads in acutely ill subjects compared with asymptomatic children might have further strengthened the association between hrv/ent and severe disease as rhinoviruses are also commonly identified in community-based controls. , future research should measure viral loads and use sequencing for genotyping analysis to differentiate hrv from enterovirus and to speciate hrv. recent studies suggest that human rhinovirus species a and c (hrv-a and hrv-c) may be associated with greater clinical severity compared with hrv-b species. , , finally, our estimation of bacterial coinfection may have been underestimated as adequate respiratory samples such as bal are rarely performed in children and pneumonia rarely result in positive blood cultures. also, urt samples are not necessarily representative of lrt disease. this limitation inherent to studies assessing the severity of respiratory illnesses in children may be overcome in future prospective studies, conducted among children with specific underlying comorbidities (e.g., immunocompromised children), which would include validated molecular assays for detection of both viral and bacterial pathogens from urt samples in all children with arti. in conclusion, our findings provide new insight into the burden and severity of hrv/ent infections and reinforce the need for routine diagnosis in hospital settings. hrv/ent infections were very common and associated with more severe disease than other common viruses such as flu or rsv highlighting the need for development and testing of specific antiviral drugs. human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children role of rhinovirus c respiratory infections in sick and healthy children in spain a novel group of rhinoviruses is associated with asthma hospitalizations association of rhinovirus infection with increased disease severity in acute bronchiolitis rhino/enteroviruses in hospitalized children: a comparison to influenza viruses viral pneumonia clinical features of patients with acute respiratory illness and rhinovirus in their bronchoalveolar lavages community study of role of viral infections in exacerbations of asthma in - year old children role of viral respiratory infections in asthma and asthma exacerbations viruses in asthma exacerbations role of rhinovirus in hospitalized infants with respiratory tract infections in spain respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants characterization of pandemic influenza a (h n ) virus specimens with a positive hemagglutinin signal in the luminex xtag respiratory viral panel assay evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: update by the infectious diseases society of america infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis guidelines for the management of adult lower respiratory tract infections induced sputum in the diagnosis of childhood community-acquired pneumonia population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children editorial response: rhinovirus pneumonia-a clinical entity? recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients community controls were preferred to hospital controls in a case-control study where the cases are derived from the hospital association between human rhinovirus c and severity of acute asthma in children acute lower respiratory tract infection novel human rhinoviruses and exacerbation of asthma in children detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study in-kind contribution of resplex ii v . kits was provided by qiagen. dr susan richardson has received honoraria from qiagen and luminex molecular diagnostics for a study of multiplex respiratory pcr in children. the funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. dr. health sciences foundation (jack hirsh fellowship). the authors had no financial or other forms of conflict of interests. key: cord- -myjyw ei authors: longtin, jean; marchand-austin, alex; winter, anne-luise; patel, samir; eshaghi, alireza; jamieson, frances; low, donald e.; gubbay, jonathan b. title: rhinovirus outbreaks in long-term care facilities, ontario, canada date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: myjyw ei diagnostic difficulties may have led to underestimation of rhinovirus infections in long-term care facilities. using surveillance data, we found that rhinovirus caused % ( / ) of respiratory outbreaks in these facilities during months in . disease was sometimes severe. molecular diagnostic testing can differentiate these outbreaks from other infections such as influenza. diagnostic diffi culties may have led to underestimation of rhinovirus infections in long-term care facilities. using surveillance data, we found that rhinovirus caused % ( / ) of respiratory outbreaks in these facilities during months in . disease was sometimes severe. molecular diagnostic testing can differentiate these outbreaks from other infections such as infl uenza. r espiratory tract illnesses are a major cause of illness and death among elderly persons, especially those in long-term care facilities. although the most commonly identifi ed viruses have been infl uenza virus and respiratory syncytial virus (rsv) ( ), human rhinovirus (hrv) is being increasingly associated with severe respiratory disease and outbreaks in these facilities ( ) ( ) ( ) ( ) ( ) . clinical diagnosis of hrv by immunofl uorescence and virus culture has been diffi cult because these methods are unreliable ( , ) . moreover, because multiple serotypes of hrv exist, retrospective serologic testing cannot be used to evaluate the prevalence of hrv disease ( ) . as a result, the number of outbreaks caused by hrv in long-term care facilities, and the associated illness and death, may be substantially underestimated. we therefore used surveillance data to estimate prevalence of hrv disease in long-term care facilities. using data from an active surveillance network, we investigated all respiratory outbreaks (as defi ned by the ministry of health) ( ), in long-term care facilities, reported from july through december , , in the province of ontario, canada. the number and timing of specimens collected was left to the discretion of the attending physicians. the regional clinical laboratories cultured specimens (blood, urine, and sputum) for bacteria and performed rapid viral antigen testing for infl uenza a/b and rsv. to facilitate turnaround time during periods of higher demand, we used an alternate multiplex nat kit (seeplex rv; seegene usa, rockville, md, usa) in conjunction with the luminex assay. because the luminex assay cannot differentiate between ent and hrv, we used the seeplex rv kit, which can identify hrv, to confi rm results in a random subset of ent/hrv-positive samples. to type the hrv implicated in outbreaks during which deaths occurred, we amplifi ed and sequenced the hypervariable region of the ′ noncoding region, the entire viral capsid protein (vp) gene, and the ′ terminus of the vp gene; we then constructed phylogenetic trees as described ( , ) . during the surveillance period, respiratory disease outbreaks in long-term care facilities were reported to the ontario public health laboratory; we received samples from facilities (table ) . a total of specimens were tested (average . samples/outbreak). of the ( %) outbreaks for which a pathogen was identifi ed, ( %) pathogens were determined to be ent/hrv (representing positive samples) and were temporally spread throughout the surveillance period. pandemic (h n ) virus and parainfl uenza- virus represented % and %, respectively, of identifi ed pathogens. other viruses were identifi ed for < % of outbreaks. viral co-infection was identifi ed in samples from outbreaks. a subset of samples, representing % of ent/hrv outbreaks, were randomly selected and subsequently tested with the seeplex rv kit to further differentiate ent from hrv; hrv was detected in % of these specimens. deaths were potentially associated with ent/hrv in facilities (outbreaks a-d; table ). samples from patients involved in these outbreaks were confi rmed to contain hrv; no other causative bacteria or viruses were identifi ed. clinical data were available for of of the patients who died; deaths were attributed to pneumonia/ respiratory infection. of the patients who died, ( %) had osteoarthritis, ( %) had cardiovascular conditions, ( %) had dementia, ( %) had diabetes, and ( %) had cancer. the only postmortem lung tissue specimen collected was positive for hrv-c (outbreak d). nucleotide sequences obtained from isolates from outbreaks a, b, c, and d showed homology to hrv-a ( %), hrv-a ( %), hrv-a ( %), and hrv-c n ( %), respectively. we performed multiple sequence alignments of the bp of the ′ untranslated region, vp /vp , and vp and compared them with published representative hrv sequences. we could not obtain a vp sequence from strains isolated during outbreak d. phylogenic trees were constructed, and the vp / vp region tree showed better discriminatory power than did that of the ′ untranslated region (figure) . vp /vp sequence identity was > % within each outbreak. sequences were deposited in genbank under accession nos. gu -gu . we cautiously assume that hrv was the causative organism for ( %) of the respiratory outbreaks in long-term care facilities in ontario during the surveillance period. multiplex molecular methods were crucial for rapid identifi cation of the pathogens involved in these outbreaks. we were able to provide results in a timely fashion for ev-ery outbreak. however, the cost and expertise associated with such technology might be beyond the reach of some clinical laboratories. because of the limitations of the sur- figure. neighbor-joining phylogenetic tree of human rhinoviruses (hrv) isolated from respiratory disease outbreaks with associated deaths in long-term care facilities, ontario, canada. tree was constructed by using a -bp nt region encoding viral capsid protein (vp) /vp , along with strains representative of hrv species a, b, and c. echo is the outgroup. bootstrap analysis used , pseudoreplicate datasets. scale bar represents . % of nucleotide changes between close relatives. boldface indicates sequences deposited in genbank. veillance program, we were unable to assess whether such testing is cost-effective in terms of patient care. of the outbreaks with associated deaths, were attributed to hrv-a and to hrv-c. the link between respiratory disease severity and hrv-c speciation is debatable ( ) . in a study from hong kong, ( %) of adults with hrv-c infection had pneumonia compared with ( %) of adults with hrv-a infection ( ) . however, in the cases we studied, most deaths were associated with hrv-a; a recent study found that hrv-c disease had the same indistinct clinical presentation as did other hrv diseases ( ) . viruses isolated from nasopharyngeal swabs by sensitive nat may represent asymptomatic colonization or nonliving organisms. although postmortem specimens were available for analysis from only outbreak-related case, we identifi ed hrv in the postmortem lung specimen. because we do not know whether hrv was present in the lower respiratory tract of the remaining patients who died, a causal association between hrv and severe disease must be made cautiously. we used the nat assays interchangeably because their reported specifi city is > % for all targets ( ) . sensitivity for each assay differs according to target; compared with the luminex assay, seeplex rv is more sensitive for parainfl uenza, rsv, coronavirus, and adenovirus but less sensitive for hrv ( ) . however, despite limitations for epidemiologic data collection, no pathogens other than hrv could explain these outbreaks and associated deaths. our testing panel did not include human bocavirus or infl uenza c virus, which could be involved in the remaining % of outbreaks that had no identifi ed cause and could even represent confounding factors in the causal relationship of a supposed pathogen. in conclusion, using data from a routine surveillance network, we found high prevalence of hrv during a period that encompassed the fi rst and second waves of pandemic (h n ) . these fi ndings are in accordance with the increasing knowledge that hrv outbreaks cause severe and fatal disease. mortality associated with infl uenza and respiratory syncytial virus in the united states a rhinovirus outbreak among residents of a long-term care facility risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus new molecular detection tools adapted to emerging rhinoviruses and enteroviruses human rhinoviruses: the cold wars resume appendix b: provincial case defi nitions for reportable diseases. in: infectious diseases protocol molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality sequence analysis of human rhinoviruses in the rna-dependent rna polymerase coding region reveals large within-species variation a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses comparison of commercial multiplex respiratory virus pcr assays and conventional diagnosis assays for detection of respiratory viruses and swine-origin a (h n ) infl uenza virus in children we thank julia hillan, michelle perfect, lisa penney, lindsay mccafferty, erica weir, and beth henning for their help with the epidemiologic investigations of these outbreaks.dr longtin is a medical microbiologist at centre hospitalier universitaire de québec. his research interests include the epidemiology of emerging viruses and the pharmacology of antiretroviral agents. key: cord- -e fofgxq authors: ryhal, bruce title: viral disease, air pollutants, nanoparticles, and asthma date: - - journal: bronchial asthma doi: . / - - - - _ sha: doc_id: cord_uid: e fofgxq health care providers who treat patients with respiratory disease are often asked by their patients, “what caused my asthma? and what causes my asthma suddenly to become worse?” these questions have always been difficult to answer, and moving directly to a discussion of the management of asthma is a much easier road to take. in recent years, though, enough information has accumulated about the causes of asthma that one can weave a story containing useful advice that may help patients participate in the management of their disease. and there are also recent studies that can provide answers to the questions posed by physicians who have watched in puzzlement as their previously well-controlled asthma patients have spiraled rapidly out of control. this story has been growing increasingly complex, with an ever-expanding cast of players that sometimes creates a tangled web of interactions. viral respiratory tract infections are the most common triggers of significant asthma • exacerbations. "upper respiratory tract infections" (uris) do not just involve the upper respiratory • tract. human rhinovirus (hrv), which causes the common cold, is the virus most likely to • trigger an asthma exacerbation. in contrast to the usual spring and summer temperate zone pollen season, viral infec-• tions begin to peak in the fall. the number, species, and typical course of viral respiratory tract infections that trigger • asthma vary with a person's age. both acute sinusitis and asthma exacerbations are associated with viral respiratory tract • infection and therefore antibiotics are rarely needed in uncomplicated cases. sulfur dioxide, nitrogen dioxide, ozone, and particulate matter in air pollution may • exacerbate asthma, and patients should be cautioned to stay indoors when levels of these irritants are high. indoor air pollution, especially from tobacco smoke, can be reduced with benefit to the • asthma patient. introduction health care providers who treat patients with respiratory disease are often asked by their patients, "what caused my asthma? and what causes my asthma suddenly to become worse?" these questions have always been difficult to answer, and moving directly to a discussion of the management of asthma is a much easier road to take. in recent years, though, enough information has accumulated about the causes of asthma that one can weave a story containing useful advice that may help patients participate in the management of their disease. and there are also recent studies that can provide answers to the questions posed by physicians who have watched in puzzlement as their previously well-controlled asthma patients have spiraled rapidly out of control. this story has been growing increasingly complex, with an ever-expanding cast of players that sometimes creates a tangled web of interactions. this chapter will look at how viral infections, air pollution, and possibly nanoparticles may act as causal agents of asthma. the concept of causal agent, though, has a variety of different interpretations. in general, agents may act on the respiratory tract to initiate asthma or to exacerbate it. initiation (or inception or development) of asthma refers to the start of asthma in a patient who was previously entirely free of this problem. an exacerbation (or trigger or precipitating event) means the significant and often sudden worsening of an established chronic asthmatic condition. avoidance of a proven initiating factor, if possible, could permit the primary prevention of asthma. in contrast, avoidance of triggering events will not halt the disease but only decrease the number of exacerbations in someone who already has chronic illness. in studying and treating asthma, identification of a specific trigger is usually much easier than trying to prove an initiating cause. viruses that affect asthma are acting on a complex and varied phenotype, and therefore the outcome of each infection can be quite varied. a simple linear cause-and-effect relationship between a viral infection and an asthmatic episode usually does not exist. koch's modified postulates for infection-caused disease are: the microorganism must be present in every case of the disease. • the microorganism must be isolated from a diseased organism and grown in pure • culture. the cultured microorganism should cause disease when introduced into a healthy • organism. the microorganism must be recovered from an inoculated, diseased experimental host. • this linear way of looking at viral-induced disease is not comprehensive enough to allow sufficient insight into the relationship between viral illness and asthma. no one viral infection consistently causes asthma in all or even most individuals. systems biology, though, can provide a conceptual framework for better understanding of the virus-asthma interaction. systems biology looks at the web of factors in the initial state of the individual patient and then examines how one or more external or internal influences perturb this state ( ). in fig. , the path taken by system a illustrates how one factor, for example a simple rhinovirus infection, may have very little long-term effect on mucosal inflammation in an individual with no atopic stressors and no genetic propensity toward asthma. this individual will return quickly to equilibrium and a low inflammatory state. the path of system b illustrates how multiple stressors, including genetic factors and atopic immune development, may interact with a viral infection to cause a long lasting or perhaps permanent change in the level of mucosal inflammation. some details of risk factors will be outlined and discussed in this chapter, but systems biology or systems medicine cannot yet specify each feature of the set of interactions in a way that leads to firm predictions about asthma. out of the complexity of the systems approach, though, some simple and compact principles do emerge, so that every precondition does not have to be known to predict the outcome of intervention or treatment. some general factors that appear to be important in the asthmatic response to viral infection include: though two-dimensional paper does not allow multidimensional maps, we can walk down a branching path in a narrative fashion to show the interaction of factors important in viral-caused asthma. in most of the twentieth century, the office or hospital diagnosis of viral respiratory infection was most often a good guess, a probability statement. common and more affordable viral molecular diagnostics, especially reverse transcriptase pcr (rt-pcr), and viral culture can now improve the accuracy of the guess when precision is needed. viruses may be detected in symptomatic or in asymptomatic patients. two thirds or more of acute respiratory tract infections (rtis) occurring in the community can be identified as viral. traditionally, these have been divided into upper and lower rti, but the difference between upper and lower infection seems to be more indistinct than previously believed. human rhinovirus (hrv), for example, replicates initially in the upper respiratory tract yet may cause extensive lower respiratory tract illness. the frequently used term viral upper rti (urti) is somewhat of a misnomer. the most commonly occurring respiratory virus is hrv, which accounts for nearly half of cases of viral respiratory illness, followed by influenza virus and coronavirus, with lesser contributions from parainfluenza virus, respiratory syncytial virus (rsv), adenovirus, metapneumovirus, and other miscellaneous viral species ( ) (see table ). the three main types of viruses that are known to affect asthma are hrv, rsv, and influenza. the peak periods of viral infection tend to vary from year to year, but generally in north america rhinovirus peaks in the fall and early spring, influenza in the early winter, and rsv in midwinter (fig. ) . many communities can monitor the progress of these annual epidemics with viral culture and molecular diagnostics, thereby giving physicians a higher probability of knowing in advance what virus a patient may have. a molecular diagnostic panel is commercially available for identifying acute viral respiratory infection, though the cost-effectiveness of this type of testing for routine clinical use is yet to be determined. more details of the immunobiology of the major asthmogenic viral infection, hrv, have been revealed in the past several years ( ) . the intercellular adhesion molecule icam- found on nasal epithelial cells is the attachment point for the majority of serotypes of hrv( ). hrv is divided into clades or strains hrv-a, hrv-b, and hrv-c. hrv-c has proven extremely difficult to culture. there are over different serotypes ( ). viral species influence asthma in the various age groups in different ways. age is a marker for the development and maturation of the immune system, which diversifies greatly over time. as the human body ages, the immune system molds itself to the environment to become a mirror image of specific, usually protein, molecules in the external local surroundings. age also has an important effect on the physics of scaling in the respiratory system. airway resistance is inversely proportional to the fourth power of diameter, which enlarges with age until young adulthood and then slowly declines. small increases in airway diameter therefore lead to huge reductions in airway resistance and give more "breathing room." about % of significant, prolonged wheezing episodes in children are triggered by respiratory viruses and hrv is most often involved ( ). the common rhinovirus cold accounts in large part for the fall seasonal peak of asthma in school-age children. epidemiologic evidence combined with viral molecular diagnosis has suggested that this peak is a consequence of children returning to the classroom with the subsequent spread of respiratory viruses, mainly rhinoviruses ( ) . viral exacerbations of asthma tend to be prolonged and severe. triggers such as a gust of pollen-laden breeze may be ameliorated by moving the young patient indoors, and exercise triggers can be removed by stopping the exercise, but a viral trigger is usually steady and persistent and replicates within the body. a study of children aged - years with asthma concluded that an asthma exacerbation was of a greater severity if a viral infection was present as opposed to a nonviral illness ( ) . airway hyperreactivity and a corresponding cough and wheeze may be noted for well over weeks after a rhinovirus infection in the asthmatic child. atopy confers additional risk on asthmatic children who become ill with respiratory virus infection ( ) . school-aged children with atopic asthma, as opposed to those with nonatopic asthma, have been noted in a number of studies to experience more frequent symptomatic colds, more episodes of viral-triggered asthma, and more prolonged airway hyperreactivity after the colds ( ) ( ) ( ) . the tendency to have higher numbers of symptomatic rtis and a longer duration of illness was also noted for allergic children in general, with and without asthma ( ) . parents of children with atopy and asthma tend to be frustrated by the prolonged recovery time compared with their nonatopic siblings, and school absences are more problematic. inhaled corticosteroids and leukotriene receptor antagonists (ltras) are well known to control the number of wheezing exacerbations in school-age children with chronic persistent asthma, an effect that appears to encompass those episodes caused by viral illness. a survey of school children in ontario found that children presenting in september to the emergency department for asthma exacerbations, presumably mostly viral triggered, were less likely to have used preventive anti-inflammatory medications than their counterparts who did not have such severe exacerbations ( ) . a retrospective study suggested that inhaled fluticasone and salmeterol administered prior to and during the fall could reduce the morbidity of the fall viral season in patients with asthma ( ) . a trial of a montelukast added to usual asthma therapy was able to attenuate the fall asthma peak in one study ( ) though this effect did not reach statistical significance in a later trial ( ) . inhaled corticosteroids might be expected to prevent viral-induced wheezing in children with minimal chronic disease as well. a preventive effect, though, has not been consistently shown in clinical trials. a study conducted in school-aged children without persistent disease but with a history of viral-triggered wheezing demon strated that inhaled beclomethasone diproponate was not superior to placebo in reducing future episodes. the inhaled steroid failed to reduce days with symptoms, or the frequency, severity, or duration of episodes of upper or lower respiratory illness ( ) . preventive medication should therefore be targeted especially to those patients with persistent chronic asthma. for acute treatment of a viral-provoked asthma exacerbation, oral systemic corticosteroids continue to be the most effective choice ( ) and are part of the current therapy protocols ( ). use of high-dose acute corticosteroid inhalers continues to be studied with varying success. whether vaccination can prevent asthma exacerbations is unclear. the expert panel report concluded that influenza vaccine does not reduce the frequency or severity of asthma exacerbations during the influenza season ( ). many patients in the community with asthma experience severe complications from an influenza infection, so all reasonable means of prevention should still be taken, including vaccination. the influenza virus appears to be a less potent trigger of asthma than hrv, and influenza peaks are not as well correlated with childhood asthma peaks as in the case of hrv. an oral influenza antiviral (oseltamivir) improved pulmonary function and reduced exacerbation frequency in one randomized, placebo-controlled trial in school-age asthmatic children who had influenza ( ) . unfortunately, increasing resistance of the influenza virus to antiviral agents limits their use as a long-term strategy to reduce illness in asthmatic children. the concept of using antivirals to reduce asthma morbidity in children seems theoretically promising. the preschool years can lay the groundwork for the later asthma issues of the type that have been discussed. diagnosing viral-triggered asthma in infants and preschool children, though, must be done with caution. asthma is defined as a chronic disease, and several, or even many, self-limited acute wheezing illnesses do not necessarily add up to a chronic illness. often children in this age group will experience wheezing in association with a variety of viral infections. parents are naturally anxious about treatment and prognosis in these children. preschool children who experience rsv-and hrv-induced wheezing are more likely to develop asthma in later years. the childhood origins of asthma study (coast) showed that viral wheezing illnesses in infancy and early childhood caused by hrv were the most significant predictors of the subsequent development of asthma at age ( ) . a bidirectional causation has been proposed with rsv: severe rsv was associated with a short-term increase in bronchial hyperresponsiveness, and, in turn, the presence of asthma was associated with long-term increased susceptibility for severe rsv disease ( ) . whether early childhood viral infection initiates a series of events that lead to asthma has been an area of much interest and study. one analysis showed that infants reaching months of age at the winter virus peak had a % increased risk of developing later asthma compared with those reaching age year at the winter peak ( ) . if viruses do initiate asthma in some patients, then prevention of rsv or hrv or a similar illness in a critical time period might prevent or reduce the frequency of asthma in later years. nonatopic infants who had received palivizumab (a humanized mab against rsv) for prevention of rsv infection showed an % reduction in risk of recurrent wheezing from ages to ( ) , though no effect was noted in atopic children. the hypothesis that early viral infections lead to asthma is made less convincing by epidemiologic studies showing that frequent exposure to viral rtis throughout early childhood may actually decrease the risk of later asthma. studies in the united states and in the united kingdom have shown that day care attendance and other factors that increase the frequency of viral rtis reduce the risk of later (after - years) frequent wheezing ( , ) . one interesting medical editorial on this topic was subtitled with tongue-in-cheek, "please sneeze on my child" ( ) . that strategy may not be practical, but clinicians should be able to reassure worried parents that day care exposure does not seem to result in a long-term risk of asthma. while the factors that contribute to the development of asthma are still unclear, there is little doubt that viral infections act as potent triggers of asthma in preschool children. as noted, hrv is the most potent of triggers, though all hrvs do not seem to be alike. pathogenicity of hrv appears to vary among groups a, b, and c. hrv-c was found in a study of hospitalized preschool children to be associated with asthma more often than hrv-a ( ), and hrv-c was noted to be the most frequent type found in patients presenting to the emergency department ( ) . in contrast, experimental infection with a type of hrv-a resulted in no worse illness in allergic than in nonallergic subjects ( ) . knowledge of a circulating virulent hrv strain in the community could put clinicians on the alert for more serious symptoms in their asthmatic patients with colds. there are several competing classification systems for the wheezing preschool child that aim to help with prognosis and treatment ( table ) . as a conceptual model, one can create two opposing poles. at one pole is the small child who experiences rare mild wheezing with acute viral illness, has no wheezing or cough between episodes, and has no atopy or parental asthma. these children appear to benefit very little or not at all from acute or chronic corticosteroid therapy for viral-triggered wheezing illness ( ) . at the other pole are children who wheeze daily or weekly, have an atopic history, have a parental history of asthma, and may be on chronic controller therapy. a viral infection in these children appears to be a trigger that requires a step up in asthma therapy, perhaps to a burst of oral corticosteroids. between these poles of severity are many children whose therapy must be individualized. the criteria from the national asthma education and prevention program help select preschool children who may benefit from acute and/ or chronic corticosteroids. these guidelines use the asthma predictive index ( ) to specify which wheezy small children have or likely will have chronic asthma and could benefit from various forms of inhaled and oral corticosteroid therapy. owing to concerns about oral corticosteroids, other forms of treatment for viral wheezing have been examined in preschool children. a study in -to -year-old children showed a benefit of episodic high-dose inhaled steroids with viral rti and wheezing ( ) , though some adverse effects on growth were noted. the effectiveness of a ltra, montelukast, was examined in a study of - year olds with a history of intermittent asthma. this study showed a reduction of typically viral-induced asthma exacerbations in children given the ltra as a daily controller ( ) . both inhaled corticosteroids and ltras are options to control chronic asthmatic wheezing in this age group ( ). prolonged or chronic cough after viral rti may be a problem. preschool children, whether asthmatic or not, spend a considerable percentage of the year with viral rti symptoms that are distressing to patient and parent. the years from teen through young adulthood tend to be the healthiest years of an individual's life. an expanded antiviral immunologic repertoire helps in reducing the number of annual viral rtis. while childhood is the time of most frequent viral rtis, young adults who are exposed to their own small children may have a secondary peak near their s. acute sinusitis is a common problem in this age group. sinusitis has been known to precede a worsening of asthma, and episodes of acute sinusitis have often been the occasion for a course of antibiotics. the entity of viral rhinosinusitis, though, is far more common than previously believed. a viral rti can produce a week or more of purulent discharge and radiographic abnormalities of the sinus cavities on ct scans ( ) . most acute sinusitis is not predominantly initiated by bacteria nor, at least in the first week or so, antibiotic-responsive ( , ) . the mechanism by which acute viral sinusitis becomes linked with worsening asthma is generally through the association of both diseases with viral infection (fig. ) . the adult group of patients with asthma diverges into several different phenotypes, likely representing various diseases. asthma is often said to be a syndrome rather than one disease. different phenotypes may have varying responses to viral infection. a cluster analysis divided asthma patients into five different groups. one group, "benign asthma" seemed to have well-controlled symptoms regardless of triggers, viral or otherwise. another group that was female preponderant, "obese nonesosinophilic," had minimal atopy or eosinophilic inflammation yet a high level of symptoms in response to typical triggers ( ) . chronic adult diseases of previously unknown cause have occasionally been found, in whole or in part, to have an infectious etiology. these include peptic ulcer disease (helicobacter pylori), polyarteritis nodosa (hepatitis b and c), reactive arthritis (shigella and chlamydia), and lyme arthritis (borrelia burgdorferi). a survey of asthma patients, of mean age , suggested that % of initial attacks started after an illness suggestive of a respiratory infection ( ) . this subset tended to be nonatopic and may represent a distinct phenotype. viral and nonviral initiating infectious agents have been proposed for adult "infectious asthma," including mycoplasma, chlamydia, adenovirus, and adult rsv, but reasonable proof of an infectious mechanism is still pending. regardless of initiating cause, asthma is exacerbated in adults, as in children, by viral respiratory infections. respiratory virus is found at least % of the time in adults with asthma exacerbations, but not as frequently as in childhood acute significant wheezing episodes ( ). older and elderly adults experience some degree of immune senescence but also have expanded specific antiviral immunity. the types of viral illness that exacerbate asthma are slightly different than in younger years. the peak of ed visits and asthma admissions for adults over tends not to be in the fall but rather from december to january, suggesting a broader range of viral triggers than in the fall rhinovirus peak ( ) . the contribution of influenza to excess morbidity in older adults is well known, but less generally appreciated is the contribution of rhinovirus to serious illness. concomitant heart disease, chronic obstruction pulmonary disease, and hypertension can make viralexacerbated asthma a more complicated and serious illness in older adults. a rhinovirus outbreak in a nursing home for elderly patients resulted in two thirds of the affected patients having lower respiratory tract symptoms, nearly one-third requiring corticosteroid or bronchodilator therapy, and three individuals having serious morbidity including one death ( ) . a peak of rhinovirus rti may be seen in grandparents who care for small children. consistently effective treatments for viral-caused respiratory disease have been frustratingly slow to arrive in the modern pharmacopoeia. despite these obstacles, however, a proactive approach, including vaccination and respiratory hygiene, can improve the care of the patient at risk for viral illness and bronchospasm and avert complications. since the time of albert einstein, scientists have known to be wary of "spooky action at distance." particles that affect the respiratory tract must first be dispersed into the air and enter and contact the respiratory tissue to have an effect. these particles vary in size from molecules in the angstrom range ( × − m), to so-called nanoparticles ( - × − m), to large pollen grains ( × − m), on up to the largest dust particles that can remain suspended in air (about × − m). air particles are divided into several common ranges for study purposes: • pm -particulates of an aerodynamic diameter of less than mm or , nm • fine particles of diameters below . mm or , nm • ultrafine particles or nanoparticles of diameters below . mm or nm study of the real-world, clinical effects of the individual components of air pollution is challenging since most or all components tend to be released into the air at about the same time. unwanted and/or unhealthy gases and particles make up the components of air pollution. outdoor air quality issues vary to great extent by specific location and depend on weather and climate, the level of vehicle traffic, and the type of fuel used for energy and manufacture. in the united states, the office of air quality planning and standards (oaqps) has established the national ambient air quality standard (naaqs) for each of the several pollutants. carbon monoxide, lead, nitrogen dioxide (no ), ozone, sulfur dioxide (so ), and particulate matter in the air have maximum exposure standards (fig. ) . studies of the effect of air pollution on health attempt to use statistical analysis to separate the individual contribution of each component of pollution. additionally, provocation/exposure testing can be performed in the laboratory. many of the same questions that can be asked about viral disease can be asked about air pollution -does it initiate asthma and does it trigger asthma? clearly not everyone breathing air pollution gets asthma or wheezing, but exposure does seem to increase the risk. a population study in the netherlands found that children with higher exposure to traffic-related pollutants (no , particulate matter) were more likely to develop asthma ( ) . data from the taiwan children health study showed an increased prevalence of bronchitic symptoms among children with long-term exposure to outdoor air pollutants ( ) . in addition to irritant properties, air pollution may contain immunologically active particles. nanoparticles, including particles of diesel exhaust, which are suspended in air are especially interesting to immunologists studying the development of asthma. they have been proposed to act as adjuvants and immunomodulators ( ) . most diesel particulates have sizes of less than mm and represent a mixture of fine particles and nanoparticles. acute wheezing may be triggered by exposure to high levels of pollutant gases including nitrogen dioxide, sulfur dioxide, and carbon monoxide. burning of fossil fuels is the main source of these pollutants in most locations. nitrogen dioxide and sulfur dioxide gases diffuse rapidly and impact upon the wet respiratory tract to produce highly irritating acids. sulfur dioxide can cause respiratory constriction in asthmatic patients at concentrations of . ppm when exercising ( ) . healthy adults begin experiencing increased airway resistance at ppm, and even nonasthmatic adults will develop bronchospasm at ppm, though these levels would be highly unusual in outdoor air pollution. nitrogen oxides, and especially no , are also irritating to the upper and lower respiratory tracts at low levels, and patients with asthma are more susceptible to these adverse effects. higher concentrations of outdoor no were associated with more asthma symptoms in a study of inner city children ( ) . though the mechanism of action is uncertain, exposure to carbon monoxide in city air was found in one european study to worsen lung function in adult patients with asthma ( ) . ozone, while of critical importance to global health in the upper atmosphere, is an especially noxious chemical when generated at or near ground level. ozone (o ) is not produced directly by traffic or by hydrocarbon burning. instead, the combination of no and hydrocarbons with air and sunlight form the secondary pollutant ozone. high average ozone and airborne particulate matter were associated with more frequent asthma symptoms and ed visits and hospital stays in a study of asthma sufferers in the san joaquin valley in california ( ) . ozone from air pollution has been shown to exacerbate asthma in children and adults, though this effect may be greater in children ( ) . a study of over , emergency department visits in atlanta for pediatric asthma showed a relationship to ozone and primary pollutant concentrations from traffic sources. these pollutants increased ed visits even at relatively low concentrations ( ) . the study of particulate matter in the air is quite complex, since the exact composition varies geographically. in general, high levels of particulate matter have long been associated in epidemiological studies with increased levels of respiratory disease. ongoing research is examining the importance of particle size, fine versus more coarse, in asthma and chronic respiratory illness. one study in turkey showed an % rise in asthma admissions when air contained high levels of coarse particles ( ) . in contrast, a us study did not find increased hospitalizations for respiratory disease during those periods with high coarse particle levels ( ) . the evidence for a negative effect on health from suspended fine particles is stronger ( ) . genetic and phenotype differences may be important in the sensitivity of the asthma patient to air pollution. the risk of childhood asthma in mexico city was modulated in some children by genes controlling the response to oxidative stress, such as might occur while breathing ozone ( ) . advice on how to avoid high concentrations of air pollutants is important for asthma patients. air pollution, like pollens and viruses, follows a seasonal pattern, and knowledge of the local pattern can help the primary physician with diagnosis and treatment. in the united states, a daily air quality index (aqi) is computed and distributed for most large population areas. the aqi, which is determined on the basis of the highest pollutant of the day, may be considered safe for patients with chronic respiratory disease if less than (green zone). on days with poor air quality, asthma sufferers should come inside where pollutant levels are typically much lower. indoor ozone levels vary from to % of outdoor concentrations, depending on the size of outdoor air flows into a building ( ) . although asthma patients should continue their controller therapy during periods of high air pollution, pretreatment with controller medications may not always be successful. budesonide treatment in one study was not successful in preventing ozone-triggered functional airways impairment in test subjects with mild persistent asthma ( ). while outdoor air pollution rightly receives a great deal of media and government attention, indoor air pollution can make living inside hazardous as well. fortunately, indoor air problems are usually amenable to personal control and behavioral advice. air quality issues may occur in both home and work environments. the field of occupational medicine examines workplace concerns and is reviewed in another chapter. home air quality is typically not regulated, though pollution may result from several indoor sources. hydrocarbon fuels are, of course, burned inside as well as outdoors. indoor gas cooking and heating stoves may produce no , high levels of which have been associated with increased asthma symptoms in children ( ) . good ventilation is essential if natural gas is to be burned indoors. indoor nitrogen oxides are also produced by wood-burning stoves, especially if not well vented. the most serious and prevalent type of home air pollution is secondhand or environmental tobacco smoke (ets). the risk from this indoor pollutant begins in utero. maternal smoking during pregnancy is associated with increased infant wheezing ( ) . this risk of respiratory morbidity continues to increase with postnatal parental smoking ( ) . laws regulating indoor tobacco smoking in one european country were followed by improved quality-of-life scores in a group of asthmatic indoor workers ( ) and also by a reduction in the overall rate of hospitalizations for childhood asthma ( ) . as noted previously, indoor ozone is usually much less than outdoor levels. in recent years, though, indoor ozone generators have been marketed to the public for odor control and purported heath benefits. a us epa review has warned the public about the potential hazards of adding an additional amount of a measured air pollutant to the indoor air. this chapter has examined some of the most significant initiating and exacerbating causes of asthma. viral respiratory infections, and to a lesser extent air pollution, are common triggers of exacerbations and may interact with individuals to affect the development of some forms of asthma. these causal factors do not exist in isolation but rather interact with the personal attributes of each patient, including his or her genetic and environmental background. the role of viruses and pollutants in asthma is important knowledge that has consequences for prevention, treatment, and avoidance of illness. helpful education may be given to patients and appropriate treatments selected, and health care providers can avoid the considerable human effort and resources wasted on interventions that are useless or harmful. viral and pollutant triggers demonstrate that the highly complex inflammatory asthmatic response is called forth on many more occasions than simply by the contact of pollen grains and other allergens with the respiratory tract. since so much of immune inflammation seems to have arisen from the need to combat infection, the interaction between asthmatic inflammation and viral infection is a natural topic for further investigation. some of the most significant advances in medical care have come through the treatment and prevention of viral illnesses, and furthering the understanding of respiratory viruses is a worthy priority for the future study of asthma prevention and treatment. in addition to natural harmful infectious particles, humans in recent decades have added many substances of their own creation, including the molecules and particles that constitute indoor and outdoor air pollution. control of this problem is very important for overall respiratory health. important action and advice is available for each asthma patient. by understanding and anticipating respiratory viral infections and air pollution as important causes of asthma, the health care provider can provide superior care for those who suffer from this chronic disease. thanks to albin leong md and denise ryhal bsn for helpful comments and for reviewing portions of this manuscript. the clinical applications of a systems approach a case-control study of acute respiratory tract infection in general practice patients in the netherlands the role of rhinovirus in asthma exacerbations expresssion of intercellular adhesion molecule- (icam- ) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection? common cold, uncommon variation understanding the september asthma epidemic weekly monitoring of children with asthma for infections and illness during common cold seasons duration of postviral airway hyperresponsiveness in children with asthma: effect of atopy allergic children have more numerous and severe respiratory infections than non-allergic children role of viral respiratory infections in asthma and asthma exacerbations effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial the september epidemic of asthma exacerbations in children: a search for etiology dispensing of fluticasone propionate/salmeterol combination in the summer and asthma-related outcomes in the fall attenuation of the september epidemic of asthma exacerbations in children: a randomized, controlled trial of montelukast added to usual therapy the back to school asthma study: the effect of montelukast on asthma burden when initiated prophylactically at the start of the school year national asthma education and prevention programs expert panel report : guidelines for the diagnosis and management of asthma oral oseltamivir improves pulmonary function and reduces exacerbation frequency for influenza-infected children with asthma wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the causal direction in the association between respiratory syncytial virus hospitalization and asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma the effect of respiratory syncytial virus on subsequent recurrent wheezing in atopic and nonatopic children siblings, day-care attendance, and the risk of asthma and wheezing during childhood day-care attendance, position in sibship, and early childhood wheezing: a population-based birth cohort study day care, siblings, and asthma-please, sneeze on my child a novel group of rhinoviruses is associated with asthma hospitalizations association between human rhinovirus and severity of acute asthma in children effects of allergic inflammation of the nasal mucosa on the severity of rhinovirus cold oral prednisolone for preschool children with acute virus-induced wheezing the asthma predictive index: a very useful tool for predicting asthma in young children preemptive use of high-dose fluticasone for virus induced wheezing in young children montelukast reduces asthma exacerbations in -to -year-old children with intermittent asthma computed tomographic study of the common cold effect of amoxicillin-clavulanate in clinically diagnosed acute rhinosinusitis antibiotics for acute maxillary sinusitis cluster analysis and clinical asthma phenotypes infectious asthma: a reemerging clinical entity? epidemiology of asthma exacerbations a rhinovirus outbreak among residents of a long-term care facility traffic-related air pollution and the development of asthma and allergies during the first years of life air pollution and prevalence of bronchitic symptoms among children in taiwan the immune effects of naturally occurring and synthetic nanoparticles carbon monoxide pollution is associated with decreased lung function in asthmatic adults medical management guidelines for sulfur dioxide (so ): agency for toxic substances and disease registry acute respiratory health effects of air pollution on children with asthma in us inner cities outdoor air pollution and uncontrolled asthma in the relationship between visits to emergency departments for asthma and ozone exposure in greater short-term associations between ambient air pollutants and pediatric emergency department visits particulate matter (pm( . ), pm( - . ), and pm( ))and children's hospital admissions for asthma and respiratory diseases: a bidirectional casecrossover study coarse particulate matter air pollution and hospital admissions for cardiovascular and respiratory diseases among medicare patients epidemiological evidence of effects of coarse airborne particles on health nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase and glutathione s-transferase m polymorphisms and childhood asthma indoor ozone: north carolina public health epidemiology budesonide reduces neutrophilic but not functional airway response to ozone in mild asthmatics health effects of indoor nitrogen dioxide and passive smoking on urban asthmatic children international study of wheezing in infants:risk factors in affluent and non-affluent countries during the first year of life interactive effect of family history and environmental factors on respiratory tract-related morbidity in infancy respiratory symptoms, pulmonary function, and markers of inflammation among bar workers before and after a legislative ban on smoking in public places smoke-free legislation and hospitalizations for childhood asthma asthma and wheezing in the first years of life definition, assessment, and treatment of wheezing disorders in preschool children: an evidence-based approach key: cord- -bnjkmegh authors: yuan, lijuan; saif, linda j title: induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model date: - - journal: vet immunol immunopathol doi: . /s - ( ) - sha: doc_id: cord_uid: bnjkmegh enteric viruses are a major cause of diarrhea in animals and humans. among them, rotaviruses are one of the most important causes of diarrhea in young animals and human infants. a lack of understanding of mechanisms to induce intestinal immunity and the correlates of protective immunity in neonates has impaired development of safe and effective vaccines against enteric viruses. studies of candidate vaccines using an adult mouse model of subclinical enteric viral infections often do not predict vaccine efficacy against disease evaluated in neonatal large animals. a series of studies have been conducted using a neonatal gnotobiotic pig model of rotavirus infection and diarrhea to identify correlates of protective immunity and to evaluate traditional and novel vaccine approaches for the induction of mucosal immune responses and protection to enteric viruses. gnotobiotic pigs recovered from infection with virulent wa human rotavirus (hrv) (mimic natural infection) had high numbers of intestinal iga rotavirus-specific primary antibody-secreting cells (ascs) and memory b-cells (to recall antigen) measured by elispot assay, which correlated with complete protection against rotavirus challenge. most short-term iga memory b-cells were resident in the ileum, the major site of rotavirus replication. spleen, not the bone marrow, was the major resident site for longer-term igg memory b-cells. candidate rotavirus vaccines evaluated in pigs for their ability to induce intestinal or systemic asc and protection against rotavirus infection and diarrhea included attenuated live virus, inactivated virus, and baculovirus-expressed double-layered rotavirus-like particles ( / -vlps). in combination with those candidate vaccines, various adjuvants, delivery systems, and immunization routes were tested, including incomplete freund’s adjuvant for i.m. immunization, and a mutant escherichia coli heat labile enterotoxin r g (mlt) for i.n. immunization. it was shown that orally administered replicating vaccines were most effective for priming for intestinal iga asc and memory b-cell responses, but i.n. administered non-replicating / -vlps plus mlt were effective as booster vaccines. we conclude that protective immunity depends on the magnitude, location, viral protein-specificity, and isotype of the antibody responses induced by vaccination. therefore highly effective enteric viral vaccines should: (i) induce sufficient levels of intestinal iga antibodies; (ii) include viral antigens that induce neutralizing antibodies; and (iii) require the use of effective mucosal adjuvants or antigen delivery systems for non-replicating oral or i.n. vaccines. enteric viruses infect and damage intestinal epithelial cells leading to diarrheal disease (saif, b) . enteric viral infections remain a major cause of morbidity and mortality for neonatal farm animals and children (saif and jackwood, ) . a lack of understanding of mechanisms to induce intestinal immunity and the correlates of protective immunity in neonates has impaired development of safe and effective vaccines against enteric viruses for farm animals and children. protection induced by candidate vaccines observed in studies using lab animals, such as mouse and rabbit models of rotavirus subclinical infections often do not predict the protective ef®cacy of these vaccines against clinical infections in studies of neonatal large animals o'neal et al., ; mcneal et al., a,b; yuan et al., yuan et al., , welter and welter, ) . efforts to identify immunologic correlates of protective immunity to rotavirus infection using knockout mice for selected immune-related genes, passive lymphocyte transfer experiments, and immunodepletion of selected populations of lymphocytes by the injection of speci®c monoclonal antibodies have shown that none of the known effectors for acquired immune responses (cd or cd t-cells, antibodies, interferon-g, perforin, etc.) are essential for protective immunity in adult mice (franco and greenberg, ; o'neal et al., ) . the outcome of vaccine ef®cacy studies using the adult mouse model of rotavirus infection also largely depends on the genetic background of the mouse strain (franco and greenberg, ) . uncharacterized innate immune mechanisms were suggested to play a major role in protective immunity against rotavirus infection in some mouse strains (choi et al., ; franco and greenberg, ; mcneal et al., b) . the uncertainty of the roles of known immunologic effectors in protective immune responses to rotavirus in adult mice raises questions of the relevance of studies using adult mice as models for rotavirus immunity in neonatal farm animals and human infants. studies of mucosal immune responses and protective immunity induced by experimental rotavirus infections and candidate vaccines using a gnotobiotic pig model of rotavirus diarrhea (chen et al., ; yuan et al., yuan et al., , yuan et al., , yuan et al., , a or trans-missible gastroenteritis coronavirus (tgev) infection in conventional piglets (vancott et al., (vancott et al., , have consistently demonstrated that protective immunity against diarrhea from enteric viral infections depends upon the production of intestinal iga antibody-secreting cell (asc) and memory b-cell responses at the sites of virus replication (saif, (saif, , a . studies of natural rotavirus infections in children showed a similar positive correlation of iga antibodies and protective immunity against rotavirus infection and diarrhea (matson et al., ; velazquez et al., ) . strategies to enhance production of virus-speci®c iga antibodies to the neutralizing viral antigens in the small intestine (intestinal lamina propria) may be the most effective approach to induce protection against viral diarrheas. this paper will: (i) review vaccination strategies for induction of mucosal immune responses and protection against enteric viral infections and disease with emphasis on candidate rotavirus vaccines evaluated in a gnotobiotic pig model of rotavirus disease; (ii) discuss comparative vaccine protective ef®cacies evaluated using the neonatal gnotobiotic pig model of rotavirus disease versus the adult mouse model of rotavirus infection; (iii) compare the distribution pattern of memory b-cells in pigs to that of t-and b-memory cells in mice and their role in the protective immunity; and (iv) brie¯y summarize the features of three novel mucosal vaccine delivery systems (coronavirus derived expression systems, microencapsulation and immune stimulating complexes (iscoms)) and their potential for improving the ef®cacy of enteric virus vaccines. the characteristics of the mucosal immune system and the pathogenesis of enteric viruses, including rotavirus have been reviewed (mowat and viney, ; saif, b) . using neonatal gnotobiotic pigs, a unique model to study rotavirus immunity , a series of studies have been conducted to identify the immune correlates of protective immunity to rotavirus infection and disease (chen et al., ; ward et al., b; yuan et al., yuan et al., , yuan et al., , yuan et al., , a and to evaluate various vaccination approaches (vaccine types, immunization routes and adjuvants) for their ability to induce intestinal and systemic immune responses and protection against rotavirus challenge (ward et al., b; yuan et al., yuan et al., , yuan et al., , yuan et al., , b . the questions addressed were: what are the basic parameters of the host mucosal and systemic antibody responses (isotype and tissue distribution) to infectious rotavirus (virulent or attenuated) or non-replicating (inactivated or vlp) vaccines in naõ Ève pigs? how do these responses correlate with protection? how does the type of vaccine and route (oral, i.m., i.n., or sequential combinations of oral and i.n.) of administration in¯uence the mucosal and systemic antibody responses to rotavirus and the degree of protection? we ®rst investigated the asc and memory b-cell responses to a virulent human rotavirus (hrv) wa strain (to mimic natural infections) and an attenuated wa hrv strain (to mimic live oral vaccines), the degree of protection induced against virulent wa hrv challenge, and potential correlates of protection (yuan et al., (yuan et al., , a . four groups of gnotobiotic pigs were orally inoculated with one dose of virulent wa hrv at ± days of age, or or doses of attenuated wa hrv (atthrv Â, Â) at -day intervals, or mock-infected with cell-culture¯uids, respectively, and challenged at ± postinoculation days (pids) with virulent wa hrv. the virus-speci®c asc and memory b-cell responses in the intestinal (duodenum, ileum, and mesenteric lymph nodes) and systemic (spleen, peripheral blood, and bone marrow) lymphoid tissues were enumerated using an isotype-speci®c elispot assay at pid ± and postchallenge days (pcds) (pid ± ). to quantitate virus-speci®c asc, an elispot assay was performed on the day of mononuclear cells (mncs) extraction (yuan et al., ) . to detect virus-speci®c memory b-cells, an elispot assay was performed after the mnc from each tissue had been stimulated in vitro with semipuri®ed wa hrv antigen in cell culture for days (yuan et al., a) . the asc and memory b-cell responses in three representative tissues at pcd and protection rates against challenge are summarized in table . pigs recovered from virulent rotavirus infection were completely protected from virus shedding upon challenge. these pigs developed signi®cantly higher numbers of virus-speci®c iga and igg asc and memory b-cells in the intestinal lamina propria compared to the attenuated wa hrv inoculation groups. oral inoculation of pigs with or doses of attenuated wa hrv induced lower numbers of iga and igg asc and memory b-cells in the intestinal lamina propria, but higher numbers of memory b-cells in the spleen compared to the virulent wa hrv inoculated pigs. the attenuated wa hrv inoculated pigs were partially protected against virus shedding and diarrhea after challenge ( table ). the protection rates from the virulent, atthrv Â, and atthrv  inoculation groups were positively correlated with the magnitude of iga asc and memory b-cell responses in the intestinal lamina propria. a positive correlation between intestinal iga antibody responses and protection was also shown in a study of mice comparing the differences in the mucosal and systemic immune responses generated by oral inoculation of mice with heterologous (non-murine) and homologous (murine) rotaviruses and the ability of these infections to produce protective immunity against subsequent reinfection upon challenge (feng et al., ) . sucking mice were inoculated orally with live rhesus (rrv), bovine (ncdv), or murine (ehp) rotavirus and challenged at pid with virulent murine rotavirus (ecw). conclusions from this study using the adult mouse model of rotavirus infection agreed with the ®ndings from studies of pigs infected with virulent or attenuated wa hrv and challenged with virulent wa hrv; i.e., the ability of a heterologous rotavirus to stimulate a detectable intestinal iga antibody response correlated with its ability to generate protective immunity. due to the nature of enteric virus infections (super-®cial infections of the intestinal epithelial cells), iga antibodies present in the intestine at the time of exposure provide the most important ®rst line of protection by neutralizing virus infectivity in the lumen or intracellularly (burns et al., ) , preventing the viruses from attaching to and penetrating the yuan et al. ( yuan et al. ( , a . b not determined. epithelium, and excreting bound antigens (immune complexes) through the epithelium back into the lumen (saif, a) . to stimulate intestinal iga antibody responses, oral immunization is preferable because it mimics the route of natural infection. however, oral vaccines have shown insuf®cient or inconsistent ef®cacy in the ®eld and clinical trials (bresee et al., ; saif and jackwood, ) . the reduced immunogenicity of the vaccine strain compared to the virulent strain of the virus (yuan et al., ; ward et al., b) , the potential risk of adverse effects of high vaccine doses or live vaccines in infants (intussusception) (anonymous, ) , and the interference of preexisting antibodies in the intestine of neonates (mainly maternal antibodies derived from colostrum and milk) with replicating virus vaccines parreno et al., ) are major obstacles to improving the ef®cacy of live oral rotavirus vaccines. to circumvent the obstacles for oral live vaccines, non-replicating vaccines have been evaluated for their immunogenicity and protective ef®cacy. studies of adult mice and rabbits have shown that non-replicating rotavirus vaccines (inactivated virus or vlps) administered via extraintestinal immunization routes (i.m., i.n. or i.p.) induced complete or signi®cant partial protection against infection with or without adjuvants (conner et al., ; mcneal et al., mcneal et al., , mcneal et al., , a of®t and dudzik, ; o'neal et al., o'neal et al., , . we evaluated the immunogenicity and protective ef®cacy of non-replicating rotavirus vaccines in combination with different adjuvants, including inactivated wa hrv given by the oral or i.m. route with incomplete freund's adjuvant (ifa) (yuan et al., ) and / -vlp vaccines given by the i.n. route with or without a mutant escherichia coli heat labile enterotoxin r g (mlt) as a mucosal adjuvant in gnotobiotic pigs (yuan et al., ) . the mlt and other lt or cholera toxin (ct) derivatives have been shown to retain the adjuvant effects of the native lt or ct to enhance immune responses to unrelated antigens when coadministered orally or i.n. through induction of both th -and th -type immune responses (lt) or by promoting th -type immune responses (ct) (freytag and clements, ; pizza et al., ) . the asc and memory b-cell responses and the protection rates induced by the nonreplicating rotavirus vaccines are summarized in table . binary-ethyleneimine inactivated wa hrv administered orally alone or by the i.m. route with ifa twice to gnotobiotic pigs induced few iga asc and memory b-cells in the intestinal lamina propria and conferred minimal protection ( ± %) against challenge. substantially higher numbers of igg asc and memory b-cells were induced in the systemic lymphoid tissues (spleen and peripheral blood) by i.m. inoculation of pigs with inactivated wa hrv; however, igg asc or memory b-cells in the systemic lymphoid tissues were not associated with protective immunity. thus, inactivated rotavirus vaccines administered via ether the oral or i.m. route were signi®cantly less ef®cacious in inducing intestinal iga antibody responses and conferring protective immunity than the oral live rotavirus vaccines. the segregation of the mucosal and systemic immune responses appears to be the cause of the failure of i.m. vaccination to induce intestinal immune responses. it was shown in a study of humans that t-and b-cells activated by the oral and parenteral routes differ markedly with regard to their respective homing potentials (kantele et al., ) . the majority of lymphocytes activated by parenteral inoculation expressed l-selectin, the principal peripheral lymph node homing receptor; and the majority of this population of b-cells were committed to igg production (quiding-jarbrink et al., ) , which explains the high numbers of igg asc and memory b-cells detected in the spleen of pigs inoculated i.m. with inactivated rotavirus ( table ). the observations on the isotype and tissue distribution of antibody responses and protection in pigs inoculated i.m. with inactivated rotavirus concur with the ®ndings from studies of mice showing a relationship between the homing receptor expression pattern of memory b-cells and protective immunity rott et al., ; williams et al., ) whereby protective immunity for an intestinal pathogen, such as rotavirus, resides in the subsets of memory b-and t-cells expressing the intestinal homing receptor a b , and this phenotype is not shared with memory cells elicited by i.m. immunization. lymphocytes sensitized in the nasal-associated lymphoid tissues (nalts) can relocate to distant effector sites through the common mucosal immune system (brandtzaeg et al., ) . the advantages of nalt as an inductive site for vaccine antigens, particularly yuan et al. ( yuan et al. ( , . for non-replicating antigens, include the presence of antigen-retaining crypts and the absence of antigendegrading digestive enzymes. although the i.n. route has been shown to be more ef®cient for the induction of iga antibody responses in the upper respiratory and genitourinary tract, and less ef®cient for the intestinal lamina propria in rodents and pigs (brandtzaeg et al., ; mcdermott and bienenstock, ; vancott et al., ) , the i.n. vaccination route has been shown to be effective for induction of protective immunity against rotavirus infection in adult mice (o'neal et al., (o'neal et al., , mcneal et al., b) . in gnotobiotic pigs, double-layered / -vlp vaccines derived from co-expression of the bovine rf core protein vp gene and the inner capsid protein vp gene of simian sa or human wa rotavirus strains were evaluated using the i.n. route (yuan et al., ) . the vp core and vp inner capsid rotavirus proteins did not induce virus neutralizing antibodies which are induced only by the rotavirus outer capsid proteins vp and vp . four groups of gnotobiotic pigs received ug per dose sa / -vlps in or i.n. dose regimens (sa / -vlp mlt in  and Â), two doses of sa / -vlps alone (sa / -vlps in Â), or mlt alone as controls, respectively. intranasal inoculation of pigs with sa / -vlps plus mlt induced higher numbers of intestinal iga and igg asc and memory b-cell responses than three oral doses of live attenuated wa hrv, but lower iga and igg asc and memory b-cells in the spleen (tables and ). the mlt adjuvant enhanced intestinal asc and memory b-cell responses to the vlp vaccines, as evident by the greatly increased numbers of intestinal iga and igg asc and memory b-cells in the pigs vaccinated with sa / -vlps mlt compared to the sa / -vlps alone at pid (pcd ). however, sa / -vlps administered i.n. in two or three dose regimens with or without mlt did not confer protection to pigs against diarrhea upon challenge ( table ). the failure of / -vlp vaccines suggests that protective immunity to rotavirus diarrhea in pigs depends not only on the location, the magnitude, and the isotypes of antibodies, but also on the protein-speci®city (vp and vp neutralizing antibodies) of the b-cell responses. although a similar sa / -vlp vaccine conferred complete protection against infection when adult mice were inoculated i.n. (o'neal et al., ) , protection against diarrhea in neonatal pigs probably requires the presence of iga neutralizing antibodies in the intestine to the outer capsid rotavirus proteins, vp and vp . most non-replicating antigens (e.g. inactivated virus, recombinant proteins, and vlps) are usually poor immunogens for inducing mucosal immune responses when administered without adjuvants, and may even result in immunological tolerance instead of immunity (czerkinsky et al., ) . to induce suf®cient levels of immune responses, an effective mucosal adjuvant or delivery system that can greatly enhance the immunogenicity of the vaccine antigens is of critical importance. however, in a study of mice inoculated i.n. with puri®ed inactivated double-or triple-layered rotavirus particles, similar rates of protection were induced by the vaccines with or without adjuvant; although mlt signi®cantly increased antibody responses and reduced antigen concentrations needed for full protection (mcneal et al., b) . the discrepancies in rotavirus vaccine ef®cacies in studies of adult mice and neonatal pigs will be discussed further in a later section. gut-associated lymphoid tissues (galts) and nalt are two major inductive sites for mucosal immune responses. the use of combined oral and i.n. vaccination routes stimulates multiple mucosal inductive sites, i.e., both galt and nalt in contrast to the individual oral or i.n. vaccination route alone. we postulated that by exploiting the advantages of multiple mucosal immunization routes (oral and i.n.), and vaccine types (replicating virus and non-replicating vlps), a combined vaccination regimen may optimally stimulate the mucosal immune system and increase the protective ef®cacy of rotavirus vaccines. two combined vaccine regimens were evaluated in gnotobiotic pigs. in the ®rst combined vaccine regimen group, gnotobiotic pigs were inoculated with one oral dose of attenuated wa hrv at ± days of age to prime the galt inductive sites by live replicating virus. this was then followed by two i.n. boosting doses of wa / -vlp plus mlt (atthrv/vlp Â) at -day intervals. in the second combined vaccine regimen group, pigs were inoculated with two i.n. doses of wa / -vlp plus mlt followed by one oral dose of live attenuated wa hrv (vlp Â/atthrv Â). a single oral dose of attenuated wa hrv (atthrv Â) and three i.n. doses of wa / -vlp plus mlt (wa / -vlp mlt Â) groups were included as controls. the asc responses and protective ef®cacy of the combined regimens are summarized in table . the atthrv/vlp  regimen induced similar mean numbers of intestinal iga asc as induced after virulent wa hrv infection ( versus asc per  mnc, respectively, tables and ) and the iga asc numbers were signi®cantly higher than those induced by a single oral dose of attenuated wa hrvor three i.n. doses of wa / -vlp plus mlt at pid (pcd ). the atthrv/vlp  regimen induced the highest mean numbers of intestinal iga asc at challenge among all rotavirus vaccines tested to date in gnotobiotic pigs including one to three oral doses of live attenuated wa hrv, two or three i.m. doses of inactivated wa hrv with ifa, and two or three i.n. doses of sa or wa / -vlps with or without mlt (yuan et al., (yuan et al., , (yuan et al., , (yuan et al., , a . these results suggest that the combined immunization routes, oral priming followed by intranasal boosting, and vaccine types, live attenuated wa hrv for priming followed by / -vlps plus mlt for boosting, was more effective in stimulating intestinal iga asc responses than each individual vaccine. the protective ef®cacy of the atthrv/vlp  regimen was slightly lower than that of three doses of attenuated wa hrv (table ) with a % protection rate against virus shedding and a % protection rate against diarrhea upon challenge of pigs with virulent wa hrv. although the atthrv/vlp  regimen did not confer a higher rate of protection compared to the atthrv  vaccine, the potential advantage of the combined atthrv/vlp  vaccine was indicated by the signi®cantly higher asc responses induced in the intestinal lymphoid tissues and the potential greater safety of using a non-replicating vaccine as boosters to reduce the risk of intussusception associated with a live oral reassortant hrv vaccine in infants (anonymous, ) . the absence of the outer capsid vp and vp rotavirus neutralizing antigens from the i.n. boosting doses of / -vlps in the atthrv/vlp  regimen was again presumably a determinant in its failure to confer higher protection rates as it was for the complete failure of i.n. / -vlp vaccines tested in pigs to confer any protection. if a triple-layered / / / -vlp vaccine were used for boosting in the combined regimen, a much higher protection rate might have been achieved based on the high numbers of iga asc in the intestinal lymphoid tissues induced by this vaccine approach and based on the positive correlation between the numbers of iga asc in the intestinal lymphoid tissues induced by live rotavirus and the degree of protection (yuan et al., (yuan et al., , a . the vlp Â/atthrv  regimen was less ef®cacious than the atthrv/vlp  regimens in inducing intestinal asc responses and protection against virus shedding or diarrhea, but more ef®cacious than the i.n. wa / -vlp mlt alone (table ). the ineffectiveness in inducing intestinal asc responses by the vlp Â/ atthrv  vaccine regimen was possibly partially due to the functional compartmentalization of the primary mucosal inductive site (nalt) involved in this vaccination approach, suggesting that oral priming (at the portal of infection and the site of virus replication) with live replicating vaccine is more effective for inducing intestinal iga responses. taken together, the use of a replicating vaccine to prime lymphocytes in the major inductive site (galt) followed by boosting with a non-replicating vaccine at a second mucosal inductive site (nalt) may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against enteric infections. there are numerous reports of protective immunity against rotavirus infection in adult mice induced by different types of rotavirus antigens (e.g. live or inactivated, homologous or heterologous rotavirus (feng et al., ; mcneal et al., mcneal et al., , mcneal et al., , a of®t and dudzik, ) ; recombinant rotavirus proteins or vlps choi et al., ; o'neal et al., o'neal et al., , ; and dna plasmids (chen et al., (chen et al., , herrmann et al., ) ], using various routes of inoculation (oral, i.n., i.m., i.p., or i.d.) . when comparing studies of mice, there are discrepancies in the protective ef®cacies of rotavirus vaccines possibly due to the use of different strains of in-or out-bred adult mice or murine rotaviruses. the genetic background and virulence of murine rotavirus strains used for challenge plays a critical role in the outcome of an ef®cacy study in mice (franco and greenberg, ) . the results often differ even more dramatically between studies of rotavirus vaccines in adult mice and neonatal pigs. it is dif®cult to compare data derived from studies of adult mice with neonatal pig studies due to species genetic differences, the body size, age, and in-bred (mice) or out-bred (pigs) animals, in addition to their different susceptibilities to enteric viruses or the different pathogenesis of the viruses in mice versus pigs (conner and ramig, ; saif and greenberg, ; yuan et al., ) . inoculation of mice i.m. with inactivated rotavirus or i.n. with / -vlps mlt or puri®ed inactivated native rotavirus particles induced complete protection against infection in adult mice. however, similar vaccines did not confer protection against rotavirus disease in neonatal pigs. there are many possible reasons for the discrepancies; however, the difference in the pathogenesis of rotaviruses between pigs and mice is likely one of the most critical factors. the pathogenesis of rotavirus disease in pigs, calves and probably in human infants is similar; but it differs from that in mice (conner and ramig, ; saif et al., ; saif and greenberg, ) . rotavirus infections of young pigs, calves, and human infants induces watery diarrhea, anorexia, depression, and dehydration (conner and ramig, ; saif et al., ) . pronounced histologic changes (e.g. villous atrophy as a result of virus replication and epithelial cell lysis) occur in gnotobiotic pigs infected with virulent wa hrv (ward et al., a) and similar intestinal villous atrophy has been reported for human infants (conner and ramig, ; davidson and barnes, ; saif and greenberg, ) . in mice under days of age, homologous rotavirus infection causes diarrhea and lethargy, but only mild intestinal lesions consisting of vacuolization of villous tip epithelial cells with little or no villous atrophy (little and shadduck, ) . in adult mice, rotavirus infection occurs without causing disease or histopathologic changes (ward et al., ) . high levels of intestinal iga antibodies to the neutralizing rotavirus antigens are required to prevent rotavirus disease in neonatal pigs. in contrast, protection against infection or clearance of an established infection in mice apparently can be mediated by any one of the immune effectors (iga or igg; cd or cd t-cells) or some unknown innate immune effectors. the redundancy of immune mechanisms (innate or acquired) in mice may be better developed than that in pigs or humans. such differences are very important to consider when comparing the protective ef®cacy of a vaccine evaluated in the adult mouse model of infection with the neonatal pig model of rotavirus infection and disease. vaccination induced protective immunity depends on the induction of memory b-and t-cells. to investigate the distribution and sites of residence of memory b-cells, and their role in protective immunity to reinfection against an enteric virus, we assessed the memory b-cells responses and protection in pigs inoculated with virulent and attenuated wa hrv (yuan et al., a ). short-term rotavirus-speci®c iga and igg memory b-cells were detected in the intestinal lamina propria (duodenum and ileum), mln, and spleen, but with much higher numbers in the ileum and slightly higher numbers in mln than in the other tissues at pid and . complete protection against hrv infection was associated with sig-ni®cantly higher numbers of iga and igg effector and memory b-cells in the intestinal lamina propria (especially in the ileum) of virulent hrv-inoculated pigs at challenge (table ) . our ®ndings clearly demonstrate that the short-term memory b-cells induced by virulent wa hrv reside primarily in the ileum, the major site of rotavirus replication, up to pid . our ®ndings parallel the recent ®ndings for both memory cd and cd t-cells (mackay and von andrian, ; masopust et al., ; reinhardt et al., ) that a substantial subset of memory t-cells reside in extralymphoid tissues (e.g. skin, lung, and intestinal lamina propria) and this subset of memory cells acts rapidly to execute effector activity when exposed to recall antigen. it is logical that a similar distribution pattern of memory t-and b-cells would facilitate the cognate interaction between t-and b-cells required for the successful establishment of the t-cell-dependent immune response. based on the study of cd and cd memory t-cells (masopust et al., ; reinhardt et al., ) , mackay and von andrian ( ) proposed that immunological memory could be divided into two phasesÐa short-term phase followed by a long-term phase. the short-term memory depends on the survival and activation of the extralymphoid tissue-origin memory t-cells, whereas long-term memory depends on secondary lymphoid tissue-origin memory t-cells. in gnotobiotic pigs infected with virulent wa hrv (yuan et al., a) , the numbers of iga and igg memory b-cells in all the tissues, but especially iga in the ileum declined substantially from pid to pid . signi®cant waning of iga memory b-cell responses in the intestine from pid to pid indicates a short iga b-cell memory to rotavirus infection in neonatal pigs. our ®ndings agree with previous observations that iga memory to mucosal pathogens is short-lived and requires periodic boosting to be maintained, as often occurs after repeated reexposure to endemic enteric pathogens in the environment (bernstein et al., ; saif and fernandez, ) . at pid , the spleen had the highest numbers of igg memory b-cells compared to the other tissues, including the bone marrow, suggesting that the spleen, and not the bone marrow was the major resident site for longer-term igg memory b-cells after an intestinal viral infection (yuan et al., a) . our ®ndings for the resident sites for the shortterm (up to pid ) and longer-term (at pid ) memory b-cells agree with the two-phase theory for memory t-cells (mackay and von andrian, ) . however the time scale of the two phases observed for memory b-cells in pigs orally infected with virulent or attenuated wa hrv was shorter compared to that investigated for memory cd t-cells in mice infected with vesicular stomatitis virus (masopust et al., ) , and in the latter studies the short-term phase was proposed to last for weeks to months (up to pid ) and the long-term phase supposedly lasts for years (mackay and von andrian, ) . the difference in the observed persistence of memory cells is consistent with the different lengths of protective immunity induced by local (short-term) versus systemic infections (longer-term) with viral or bacterial pathogens. according to the conventional immunological dogma based on observations in mice, bone marrow was considered to be the major site for long-term antibody production (benedetti et al., ) . in contrast to this concept, our study demonstrated that bone marrow is not a major site for iga and igg rotavirus-speci®c antibody production or for memory b-cells in pigs from pid up to at least pid after oral inoculation with hrv, as indicated by the lowest mean numbers of iga and igg memory b-cells and asc in bone marrow compared to the other tissues tested (yuan et al., a) . studies of mice also showed that no iga and igg asc or memory b-cells were detected in bone marrow after adult mice were orally inoculated with the heterologous rhesus rotavirus strain rrv from pid to pid (moser and of®t, ) . williams et al. ( ) reported that rotavirus-speci®c, facs-sorted a b iga memory b-cells adoptively transferred from donor mice seven months after oral murine ec strain rotavirus inoculation into rag- mice preferentially resided in the lamina propria with asc numbers at least -fold higher than that in bone marrow. it was shown that the memory b-cell subset responsible for the secretory iga response and protective humoral immunity to rotavirus expressed the intestinal homing receptor, a b . in conclusion, after rotavirus infection of pigs or mice, a substantial subset of memory b-cells migrate to and reside in the lamina propria of the small intestine and another major subset of memory b-cells reside in the spleen. memory b-cells resident in the lamina propria of the small intestine likely mediate protective immunity upon reinfection with rotavirus. the memory b-cells resident in the spleen may be a source for long-term antibody production (mainly igg antibody) in the circulation, but were not associated with protective immunity to reinfection with rotavirus. bone marrow was not a major site for rotavirus-speci®c antibody production or for memory b-cells in pigs and mice orally infected with rotavirus. novel delivery systems, such as use of recombinant attenuated bacteria as carriers of heterologous antigens (gentschev et al., ) , encapsulation of antigens into microspheres, or iscoms have been investigated for enhancing the immunogenicity of mucosal vaccines. the goal of these novel delivery systems is to deliver the antigens more ef®ciently to mucosal lymphocyte populations (i.e., peyer's patch) after inoculation, or to increase the persistence of antigen in lymphoid tissues at mucosal inductive sites. a newly developed coronavirus expression system (enjuanes et al., ) showed attractive features and great potential for use as a virus vector for delivery of mucosal vaccines. since coronaviruses infect the mucosal surfaces of the respiratory and/or enteric tracts, they can target the vaccines to the mucosal inductive sites like the natural infections. non-pathogenic coronavirus strains infecting most species of interest, including humans, swine, and cattle are available to develop expression systems. in addition, the tissue and species tropism of coronavirus may be manipulated through engineering the s gene which is the tropism determinant; thus the expressed vaccine antigens may target to speci®c tissues in speci®c animal species (enjuanes et al., ) . the use of the coronavirus expression system as a potential delivery vehicle for rotavirus vaccines is of interest to evaluate in the gnotobiotic pig model of rotavirus diarrhea. microencapsulation has been extensively studied for mucosal vaccination because of its attractive features (o'hagan et al., ; o'hagan, ) , including delivery of encapsulated antigens to provide protection from stomach acidity and the myriad proteases present in the gastrointestinal tract. microencapsulation has the potential to disseminate antigen to various lymphoid tissues dependent upon the size of the microsphere; therefore, manipulation of the size of the microcapsules allows targeting of vaccine antigens to speci®c sites. varying the ratio of lactide to glycolide allows antigen release over a long or short period of time. thus it may be possible to deliver a primary immunization plus a booster immunization with a single dose of vaccine. inoculation of mice orally with microencapsulated rotavirus enhanced the frequencies of virus-speci®c iga asc and the quantities of virus-speci®c iga antibodies produced in the intestinal lymphoid tissues (moser et al., ) . use of microencapsulated wa hrv in the combined atthr/ vlp  vaccine regimen may further improve vaccine ef®cacy by providing the prolonged presence of rotavirus antigen in the inductive sites of the small intestine and enhancing the homing of antigen-speci®c t-and b-lymphocytes after i.n. boosting. iscoms are another attractive approach for delivery of mucosal vaccines. the iscoms are ± nm highly structured particles composed of antigen, cholesterol, phospholopids and adjuvant quilaja saponins (rimmelzwaan and osterhaus, ) . the advantages of iscom vaccines include their potential to induce high local and systemic antibody responses and cd t-cell responses, even in the presence of pre-existing antibodies; and their capacity to induce mhc class i restricted cd ctl responses, which are important in the clearance of established virus infections (osterhaus and rimmelzwaan, ) . iscoms have been shown to induce antibody responses and activate th cells and cytolytic t lymphocytes in a number of animal species, including non-human primates and humans (sjolander et al., ) . the immune modulating effects of iscoms for an oral non-replicating rotavirus vaccine was evaluated in gnotobiotic lambs (van pinxteren et al., ) . iscoms and iscommatrix preparations enhanced virus-speci®c igg asc responses after vaccination and signi®cantly increased iga and igg asc responses in the blood after challenge compared to lambs given inactivated virus alone. this study showed that different oral iscom vaccines could induce different types of immune responses, either th -like (recombinant uk vp iscoms) or th -like (inactivated rotavirus mixed with iscom-matrix). besides the oral route, iscoms have been also evaluated for i.m. and i.p. immunization routes. intraperitoneal injection of iscoms recruited and activated many components of the innate immune system, including neutrophils, macrophages, and dendritic cells (smith et al., ) . it was suggested that iscoms act by targeting antigen and adjuvant to macrophages and/or dendritic cells (smith et al., ) . this pathway may be important to exploit for vaccine development, especially if combined with another vaccine type or vaccination route, plus a different mucosal adjuvant, such as mlt. we are currently evaluating the ef®cacy of oral or i.n. vlp iscom vaccines in the neonatal gnotobiotic pig model, alone or combined with priming by oral live attenuated wa hrv. further exploring the combination of multiple vaccination routes, vaccine types, adjuvants, and new mucosal delivery systems may lead to the optimal stimulation of the intestinal mucosal immune system and the maximal ef®cacy of mucosal vaccines against enteric viruses. withdrawal of rotavirus vaccine recommendation long-term antibodies after an oral immunization with cholera toxin are synthesized in the bone marrow and may play a role in the regulation of memory b-cell maintenance at systemic and mucosal sites induction and persistence of local rotavirus antibodies in relation to serum antibodies regional specialization in the mucosal immune system: primed cells do not always home along the same track current status and future priorities for rotavirus vaccine development, evaluation and implementation in developing countries protective effect of rotavirus vp -speci®c iga monoclonal antibodies that lack neutralizing activity enumeration of isotype-speci®c antibody-secreting cells derived from gnotobiotic piglets inoculated with porcine rotaviruses protective immunity induced by rotavirus dna vaccines protective immunity induced by oral immunization with a rotavirus dna vaccine encapsulated in microparticles antibody-independent protection against rotavirus infection of mice is stimulated by intranasal immunization with chimeric vp or vp proteins viral enteric diseases rotavirus vaccine administered parenterally induces protective immunity virus-like particles as a rotavirus subunit vaccine oral tolerance and antipathological vaccines structural and functional abnormalities of the small intestine in infants and young children with rotaviral enteritis coronavirus derived expression systems comparison of mucosal and systemic humoral immune responses and subsequent protection in mice orally inoculated with a homologous or a heterologous rotavirus isotype-speci®c antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant sa rotavirus core-like particles (clp) or virus-like particles (vlp) immunity to rotavirus infection in mice immunity to homologous rotavirus infection in adult mice bacterial toxin as mucosal adjuvants recombinant attenuated bacteria for the delivery of subunit vaccines protection against rotavirus infections by dna vaccination effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs homing potentials of circulating lymphocytes in humans depend on the site of activation pathogenesis of rotavirus infection in mice memory t cellsÐlocal heroes in the struggle for immunity preferential localization of effector memory cells in nonlymphoid tissue fecal antibody responses to symptomatic and asymptomatic rotavirus infections evidence for a common mucosal immunologic system. i. migration of b immunoblasts into intestinal, respiratory, and genital tissues active protection against rotavirus infection of mice following intraperitoneal immunization stimulation of local immunity and protection in mice by intramuscular immunization with triple-or double-layered rotavirus particles and qs- effects of different adjuvants on rotavirus antibody responses and protection in mice following intramuscular immunization with inactivated rotavirus antibody-dependent and -independent protection following intranasal immunization of mice with rotavirus particles distribution of rotavirus-speci®c memory b cells in gut-associated lymphoid tissue after primary immunization effect of water-based microencapsulation on protection against edim rotavirus challenge in mice the anatomical basis of intestinal immunity noninfectious rotavirus (strain rrv) induces an immune response in mice which protects against rotavirus challenge microparticles and polymers for the mucosal delivery of vaccines biodegradable microcapsules for oral immunization rotavirus virus-like particles administered mucosally induce protective immunity rotavirus / virus-like particles administered intranasally with cholera toxin, escherichia coli heat-lablie toxin (lt), and lt-r g induce protection from rotavirus challenge protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin a induction of virus-speci®c immunity by iscoms serum and intestinal isotype antibody responses to wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies human circulating speci®c antibody-forming cells after systemic and mucosal immunizations: differential homing commitments and cell surface differentiation markers visualizing the generation of memory cd t cells in the whole body a novel generation of viral vaccines based on the iscom matrix expression of the mucosal homing receptor a b correlates with the ability of cd memory t cells to clear rotavirus infection expression of mucosal homing receptor a b by circulating cd cells with memory for intestinal rotavirus mucosal immunity: an overview and studies of enteric and respiratory coronavirus infections in a swine model of enteric disease enteric viral infections of pigs and strategies for induction of mucosal immunity group a rotavirus veterinary vaccines rotavirus gastroenteritis enteric virus vaccines: theoretical considerations, current status, and future apporaches animal rotaviruses comparative studies of the pathogenesis, antibody immune responses, and homologous protection to porcine and human rotaviruses in gnotobiotic piglets immune responses to iscom(r) formulations in animal and primate models immune-stimulating complexes induce an il- -dependent cascade of innate immune responses isotype-speci®c antibody-secreting cells to transmissible gastroenteritis virus and porcine respiratory coronavirus in gut-and bronchusassociated lymphoid tissues of suckling pigs contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge serum antibody as a marker of protection against natural rotavirus infection and disease development of an adult mouse model for studies on protection against rotavirus pathogenesis of an attenuated and a virulent strain of group a human rotavirus in neonatal gnotobiotic pigs development of mucosal and systemic lymphoproliferative responses and protective immunity to human group a rotavirus in a gnotobiotic pig model evaluation of killed and modi®ed live porcine rotavirus vaccines in cesarean derived colostrum deprived pigs the memory b-cell subset responsible for the secretory iga response and protective humoral immunity to rotavirus expresses the intestinal homing receptor, a b systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus intranasal administration of / -rotavirus-like particles with mutant escherichia coli heat-labile toxin (lt-r g) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs short-term immunoglobulin a b cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with wa human rotavirus protective immunity and antibody-secreting cell responses elicited by combined oral attenuated wa human rotavirus and intranasal wa / -vlps with mutant escherichia coli heat-labile toxin in gnotobiotic pigs key: cord- - jodunj authors: nan title: paediatric sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: jodunj nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- -wy d cw authors: rovida, francesca; campanini, giulia; piralla, antonio; adzasehoun, kodjo messan guy; sarasini, antonella; baldanti, fausto title: molecular detection of gastrointestinal viral infections in hospitalized patients date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . . sha: doc_id: cord_uid: wy d cw gastrointestinal viral syndromes are a common cause of morbidity and mortality in humans worldwide. etiological agents include a large number of viruses encompassing several orders, families, and genera. during the period april to april , stool samples from as many patients hospitalized at the fondazione irccs policlinico san matteo of pavia exhibiting gastrointestinal syndromes were examined for the presence of rotavirus, norovirus, astrovirus, adenovirus, rhinovirus, enterovirus, parechovirus, bocavirus, coronavirus, sapovirus, cosavirus, and aichi virus using polymerase chain reaction assays. gastrointestinal viral agents were detected in ( %) patients of the analyzed. adenovirus and norovirus were the most common viruses in this cohort, while aichi virus was the only gastrointestinal agent not detected. surprisingly, rhinovirus was one of the most frequently detected viruses. however, a potential association with gastroenteritis remains to be confirmed. viral gastroenteritis is a common cause of morbidity and mortality in humans worldwide, affecting all age groups. etiological agents include a number of viruses encompassing several orders, families, and genera. viral pathogens causing acute gastroenteritis include rotavirus (rv), members of the caliciviridae family such as norovirus (nov) and sapovirus (sav), adenovirus (hadv) and astrovirus (hastv) (eckardt and baumgart, ) . viral gastroenteritis can be more severe in young children, the elderly, and immunocompromised patients. rv causes , - , deaths per year (clark and mckendrick, ) , the majority of which occur in developing countries, and it is the most severe and common cause of diarrhea in children under years of age (wilhelmi et al., ) . frequently, novs are responsible for outbreaks and sporadic cases of nonbacterial gastroenteritis in children and adults worldwide (kele et al., ) . sav is considered an important cause of gastroenteritis in children under years of age, while it is of minor importance in adults (eckardt and baumgart, ) . hastvs and enteric hadvs cause gastroenteritis, primarily in children less than years of age (dennehy, ) . human bocavirus and human coronavirus, mainly involved in infections of the respiratory tract, are also implicated in gastrointestinal infections (clark and mckendrick, ; khan and bass, ) . members of the picornaviridae family, for example, enterovirus (ev), parechovirus (hpev), aichi virus (aiv), and human cosavirus (hcosv), are causative agents of gastroenteritis (harvala et al., ; holtz et al., ) . recently, also rhinoviruses (hrv) have been detected in stool samples (harvala et al., ) . considering the highly contagious nature of these viruses, surveillance of new cases is needed for outbreak prevention and control. unfortunately, the number of agents implicated in gastrointestinal infections makes the construction of a comprehensive diagnostic panel very challenging. the present study is aimed at evaluating the circulation of gastrointestinal viruses in hospitalized patients using polymerase chain reaction (pcr) assays. overall, stool samples stored in the period april to april from as many patients ( pediatrics and adults) with gastrointestinal syndromes hospitalized at the fondazione irccs policlinico san matteo of pavia (a teaching and university hospital with , admissions, , , outpatients visits, and , emergency consultations per year) were systematically examined for the presence of gastroenteric viruses. gastrointestinal syndrome was defined as the rapid onset of or more of the following symptoms: diarrhea, vomiting, nausea, fever, or abdominal pain. in the present study, stool samples collected from patients with diarrhea during the acute phase of gastroenteritis were retrospectively analyzed. in more detail, all samples were tested by: i) real-time reverse transcriptase polymerase chain reaction (rt-pcr) for nov, rv, hastv, ev, hrv, hpev, sav, human coronavirus (hcov); ii) real-time pcr for hadv; iii) nested rt-pcr for aiv and hcosv; and iv) nested pcr for human bocavirus (hbov). the age of pediatric patients ranged from days to years (median, years), and the age of adult patients ranged from to years (median, years). this retrospective study was performed according to guidelines of the institutional review board of the fondazione irccs policlinico san matteo on the use of biologic specimens for scientific purpose in keeping with italian law (art. d.lgs / ) . viral nucleic acids were extracted from μl of % fecal suspension, using the automated extractor nuclisens® easymag™ (biomérieux, lyon, france) coextracting dna and rna in a final elution volume of μl. pcr methods were performed according to published protocols (da silva et al., ; dare et al., ; heim et al., ; kaikkonen et al., ; kapoor et al., ; kapoor et al., ; logan et al., ; lu et al., ; nix et al., ; van doornum et al., ; van maarseveen et al., ; yamashita et al., ) , with slight modifications in the reverse transcriptase protocol as well as in the adoption of real-time pcr system (applied biosystems, foster city, ca, usa) for the amplification step. in detail, μl of eluted nucleic acids were submitted to real-time rt-pcr, real-time pcr, nested rt-pcr, or nested pcr assays using the ag-path-id one-step rt-pcr kit (applied biosystems) according to manufacturer's indications. the sequence of primers and probes was not modified ( table ). the pcr thermal profile by van maarseveen et al. ( ) and by heim et al. ( ) were slightly modified (table ) without affecting sensitivity and specificity, as internally validated by parallel amplification of positive and negative samples and according the results of the quality control for molecular diagnostics (www.qcmd. org; glasgow, scotland, uk). the performance of molecular assays in comparison with commercial assays has been previously reported . purified pcr products were sequenced using the bigdye terminator cycle-sequencing kit (applied biosystem) with an abi prism dna sequencer (applied biosystem). sequences were assembled using the sequencer software, version . (gene codes corporation, ann arbor, mi, usa). nucleotide alignments were constructed using the clustalw method with mega version . software (tamura et al., ) . overall, stool samples from as many patients ( pediatrics and adults) with gastrointestinal syndromes were analyzed. the age of pediatric patients ranged from days to years (median, years), and the age of adult patients ranged from to years (median, years). gastrointestinal viruses were detected in ( . %) patients. among these, a single pathogen was diagnosed in ( . %) patients, as multiple infections in ( . %) patients, while in ( . %) patients, no viruses were detected (fig. a) . the number of patients positive for single or multiple gastrointestinal viruses is reported in fig. b . the most common gastrointestinal virus was hadv, accounting for . % ( / ) of positive samples. nov accounted for . % ( / ) of positive samples; rv, for . % ( / ); hrv, for . % ( / ); sav, for . % ( / ); ev, for . % ( / ); hastv, for . % ( / ); hcov, for . % ( / ); hbov for . % ( / ); hpev, for . % ( / ); and hcosv for . % ( / ). none of the patients ( %) tested positive for aiv infection. among the hadv positive samples, serotype was the most frequent ( / , . %), followed by serotype - -f ( / , . % each). among calicivirus, nov was the most frequent at . % ( / ), while sav was detected in . % ( / ) samples. nov gii was more frequently diagnosed ( / , . %) than gi ( / , . %). among picornavirus, hrv was the most frequently detected ( / , . %), ev was detected in . % samples ( / ), hpev accounted for . % ( / ), and hcosv corresponded to . % ( / ) of picornavirus-positive samples. multiple infections were detected in % ( / ) of patient ( fig. a and b) . most multiple infections involved hrv (n = ), hadv (n = ), rv (n = ), nov (n = ), and hbov (n = ). multiple infections involved - viruses at the same time. the most frequently detected coinfections were hadv + hrv ( / , . %). years. the relative frequency of gastrointestinal virus positivity in stool samples according to age distribution is listed in fig. . during the study period, a cumulative infection peak was reached in the winter season (between january and march) with . % ( / ) of patients testing positive for gastrointestinal viruses (fig. ) . hadv was detected throughout the year but was more frequent in october to november and january to february. hrv was present year round, except in september and february, and peaked in may ( / , . %) and january . % ( / ). rv was detected more frequently from december to march with a peak in february ( / , . %), while it decreased during the warmest period of the year. nov was absent in september, november, and december; peaked in march ( / , . %); and was present during the rest of the year. viral gastroenteritis is one of the most frequent diseases in children and adults and continues to be a significant cause of morbidity and mortality worldwide (wilhelmi et al., ) . diagnostic panels (either molecular or immunologic) should include all virus agents potentially associated with gastrointestinal syndromes but are presently largely incomplete. detection of gastrointestinal viruses is routinely performed using immunologic assays, such as the enzyme-linked immunosorbent assay (elisa) and immunochromatographic test (ict). nevertheless, these tools have limited sensitivity . in recent years, real-time pcr techniques have shown greater sensitivity and specificity than immunological assays for detection of gastrointestinal viruses (clark and mckendrick, ; rovida et al. ; van maarseveen et al. ; wilhelmi et al. ) . in an attempt to better understand the epidemiology of viral gastroenteritis in hospitalized patients, a -year surveillance study was carried out. a limitation of the study is the unavailability of data on other gastrointestinal agents (parasites, bacteria, and fungi). thus, the results of this study are useful to verify the relative frequency of different viruses in diarrheic stools, while attributing clinical significance to the virologic data would require a larger dataset. gastrointestinal viruses were detected in about one third of symptomatic patients, a percentage somewhat lower than expected. the distribution of nov, rv, sav, hastv, and hbov infections is in agreement with data reported in previous surveillance studies in developed countries (gonzález et al., ; medici et al., ; van maarseveen et al., ) . however, unlike previous results, hadv appeared to be the most frequent viral agent. this result could be related to the different sensitivity of assays used. however, monitoring hadv gastrointestinal diseases in children aged b years over a period of years in japan showed that the annual percentage of hadv infections ranged from . % to . % indicating yearly fluctuations in hadv prevalence (dey et al., ) . hadv infection in our study appeared to peak in october suggesting a potential cluster of infection. the investigation of potential local outbreaks is beyond the scope of this study. however, this type of investigation is highly needed and should be implemented. in the present study, surprisingly, hrv was one of the most frequent viruses in stools of patients with gastrointestinal syndromes. apr- may- jun- jul- aug- sep- oct- nov- dec- jan- feb- mar- apr- in addition, hrv was the most frequent virus in multiple infections. presently, the role of hrv in gastroenteritis is debated (harvala et al., ; honkanen et al., ; lau et al., ) . our data support the presence of hrvs in a significant proportion of patients with gastroenteritis and also in the absence of other gastrointestinal viruses. however, more focused studies, including the systematic detection and culturing of viral, bacterial, and parasitic agents, are needed to understand the role of hrv in stool samples. in this study, several members of the genus picornavirus were detected, including a case of hcosv gastrointestinal infection, whose clinical characteristics have been reported elsewhere . in agreement with published data (gonzález et al. ) , we found that nov was the most prevalent of calicivirus, followed by sav. in addition, nov gii was more frequently diagnosed than gi. multiple infections (involving - viruses) were detected in about one quarter of patients. these results are in agreement with a previous surveillance study in france (tran et al., ) , which reported that, in different european countries, the percentages of mixed viral infection in stool samples ranged from . to %. furthermore, we observed that multiple infections were detected more frequently in paediatric patients than in adults. the number of viral gastroenteritis was higher in winter (from january through march) than in other seasons, but hadv and hrv were detected all year round. nov infections peaked in march, and rv was detected more frequently during the winter season. in conclusion, the findings of this study show that hadv, nov, and rv are the leading cause of viral gastroenteritis in hospitalized patients. the investigation of all enteric viruses (including hastv, ev, hpev, hbov, hcov, sav, hcosv, and aiv) is needed to improve routine laboratory diagnostics and to provide additional epidemiologic data on the circulation of gastrointestinal viruses in the hospital settings. persistent human cosavirus infection in lung transplant recipient a review of viral gastroenteritis evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-pcr: different behaviors of genogroups i and ii human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays viral gastroenteritis in children prevalence seasonality, and peak age of infection of enteric adenoviruses in japan viral gastroenteritis in adults molecular epidemiology of enteric viruses in children with sporadic gastroenteritis in valencia, venezuela parechovirus in children: understanding a new infection high detection frequency and viral loads of human rhinovirus species a to c in fecal samples; diagnostic and clinical implications pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr identification of a novel picornavirus related to cosaviruses in a child with acute diarrhea human rhinoviruses including group c are common in stool samples of young finnish children aichi virus infection in children with acute gastroenteritis in finland a highly prevalent and genetically diversified picornaviridae genus in south asian children human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections comparison of an elisa and two reverse transcription polymerase chain reaction methods for norovirus detection viral infections: new and emerging detection of human rhinovirus c in fecal samples of children with gastroenteritis real-time reverse transcription pcr detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis real-time rt-pcr assay for comprehensive detection of human rhinoviruses molecular detection and epidemiology of astrovirus, bocavirus and sapovirus in italian children admitted to hospital with acute gastroenteritis detection of all known parechoviruses by real-time pcr comparison of immunologic and molecular assays for the diagnosis of gastrointestinal viral infections mega : molecular evolutionary genetics analysis (mega) software version prevalence of rotavirus, adenovirus, norovirus and astrovirus infections and coinfections among hospitalized children in northern france development and implementation of real-time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens diagnosis of viral gastroenteritis by simultaneous detection of adenovirus group f, astrovirus, rotavirus group a, norovirus genogroups i and ii, and sapovirus in two internally controlled multiplex real-time pcr assays viruses causing gastroenteritis application of a reverse transcription-pcr for identification and differentiation of aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans we thank daniela sartori for careful preparation of the manuscript and laurene kelly for revision of the english. this work was supported by the ministero della salute, fondazione irccs policlinico san matteo, ricerca corrente (grant no. ). the authors declare no competing financial interest. key: cord- -hsa r ns authors: bourdillon, n.; yazdani, s.; schmitt, l.; millet, g. p. title: effects of covid- lockdown on heart rate variability date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: hsa r ns introduction: strict lockdown rules were imposed to the french population from march to may , which may result in limited possibilities of physical activity, modified psychological and health states. this report is focused on hrv parameters kinetics before, during and after this lockdown period. methods: participants were included in this study, who underwent regular orthostatic tests (a -minute supine followed by a -minute standing recording of heart rate (hr)) on a regular basis before, during and after the lockdown (bsl, cfn and rcv, respectively). hr, power in low- and high-frequency bands (lf, hf, respectively) and root mean square of the successive differences (rmssd) were computed for each orthostatic test, and for each positions. subjective well-being was assessed on a - visual analogic scale (vas). results: out of the participants, (wb+) reported an improved well-being (i.e., increase > in vas score) during cfn, contradictory to the other participants (wb-). there was an increase in hr and a decrease in rmssd when measured supine in cfn and rcv, compared to bsl in wb-, whilst opposite results were found in wb+ (i.e. decrease in hr and increase in rmssd in cfn and rcv; increase in lf and hf in rcv). there was a moderate significant correlation between vas and hr, rmssd, hf, respectively, in the supine position; the higher the vas score (i.e., subjective well-being), the higher the rmssd and hf and the lower the hr. in standing position, hrv parameters were not modified during cfn. conclusion: the strict covid- lockdown likely had opposite effects on french population as % of participants improved parasympathetic activation (rmssd, hf) and rated positively this period, whilst % showed altered responses and deteriorated well-being. the changes in hrv parameters during and after the lockdown period were in line with subjective well-being responses. these results confirmed the usefulness of hrv as a non-invasive means for monitoring well-being and health in the general population. the covid- pandemic in resulted in strict confinement rules in france for weeks, from march to may, period known as the lockdown. its aim was to slow down the spread rate of covid- , therefore saving lives and decreasing the workload in hospitals, which capacity to treat new patients was exceeded. restrictions during the lockdown included closed gyms and sports centers, plus limitations on the duration spent outdoors, maximum walking distance from home which, associated with the lack of space and appropriate devices in homes for physical exercise, and lack of technical knowledge of the population on appropriate training routines, resulted in decreased physical activity [ ] . a survey by the french public health national institute "santé publique france" published on th june confirmed that . % of the population declared a decrease in physical activity, especially ( . %) in walking time, during the lockdown period. contradictory, a minority ( . %) used this period for increasing their physical activity. in addition, . % of the population declared spending more than h seated. the average daily seated time was h . beyond these self-declared data, the impact of the lockdown period remains unclear, but one may hypothesize that it may have led to changes in cardiovascular fitness and psychological states [ ] . exercise plays a fundamental role in cardiovascular health [ ] but also in mitigating the psychological impacts of lockdown [ ] . heart rate variability (hrv) is a commonly used method for cardiovascular follow-up in athletes [ ] . specifically, hrv analysis allows to evaluate the modulation of the sympathetic and parasympathetic branches of the autonomic nervous system. the low-frequency (lf) band reflects a mix of sympathetic and parasympathetic modulations on the heart [ ] with likely a sympathetic preponderance. the migration of the respiratory sinus arrhythmia in the low-frequency band is a subject of debate [ ] . the sympathetic modulations seen in lf are likely due to the vasomotor tone or to the central modulation of the sympathetic tone [ ] , which is linked to changes in arterial blood pressure [ ] and baroreflex activity [ ] . respiratory sinus arrhythmia is the main phenomenon provoking changes in the hf band which therefore mainly reflects parasympathetic influences on the heart [ ] . physical exercise increases the parasympathetic tone [ ] . the best performance occur with hypertonia of both the sympathetic and the parasympathetic nervous systems [ , ] . hypotonia of one or the other is a sign of cardiovascular deconditioning [ ] . finally, hrv-based methods have proven valid and reliable in the evaluation of stress, wellbeing and recovery [ ] . to the best of our knowledge, hrv parameters before, during and after the lockdown period have not been reported in the literature. therefore, the aim of the present study was to report hrv parameters to demonstrate the effects of lockdown and to investigate whether changes in hrv were related to changes in psychological states during and after this period. we hypothesized that alteration in parasympathetic-related markers (i.e., decreases in rmssd and hf, increase resting hr) would reflect a negative feeling over the period, whilst a minority of the population with a positive appreciation of this particular period would report an improved hrv. strict lockdown (further denoted cfn) rules applied in france over weeks, from march to may . the lockdown was unexpected, therefore there was no previous intention to study its effects on hrv. we used hrv monitoring setup dedicated to other studies to collect data. data collection was therefore performed for the purpose of this study. three periods were compared: from january to march (baseline, bsl); from march to may (cfn), and from to may (recovery, rcv). the local ethical committee approved the study (agreement - ; commission cantonale d'ethique de la recherche sur l'être humain, ccer-vd; lausanne, switzerland) as part of a set of studies on a broader scale. all experimental procedures conformed to the standards set by the declaration of helsinki the participants in this study were equipped with hrv monitoring devices before the beginning of lockdown. inclusion criteria were, age between and years old, physically active, healthy, non-smokers, with no known diseases and no pregnancy or lactation for women, living in france. out of the database (n = ), participants with at least forty orthostatic tests between january and may and at least tests in each period of three periods (bsl, cfn and rcv) were extracted (n = ). the participants performed an orthostatic test ( minutes supine followed by minutes in the standing position) on a regular basis between the st of january and the st of may [ ] . this orthostatic test had to be performed, in the morning, upon wake-up, with an empty bladder, and before breakfast. the inter-beat interval (rr interval) measuring device (sensor h + chest belt, polar, kempele, finland or cardiosport tp +, cardiosport, waterlooville, uk) was connected to the participants' smartphones via bluetooth (mobile application: incorpus ® v . . , be.care sa, renens, switzerland). data were immediately and automatically transferred to our servers for analysis. therefore, meetings between participants and researchers were avoided, in the respect of the strict lockdown rules applying. the rr intervals from the orthostatic tests were first analyzed to remove ectopic beats from the recordings. ectopic beats were then compensated for by means of interpolation to calculate normal-to-normal (nn) intervals. from the nn intervals, the following heart rate variability (hrv) parameters were extracted: mean hr; the root mean square of the successive differences (rmssd); the spectral power in the low-frequency (lf, . - . hz) and high-frequency bands (hf, . - . hz) in ms ; and the values (expressed in normalized units) for lf and hf, labelled nlf and nhf, respectively [ , ] . the spectral power was estimated using a fast fourier transform on the resampled nn intervals ( hz) using a window length of data points and an overlap of %. all computations were performed separately for the supine (su) and standing (st) positions using. all analyses were performed using matlab ® (r a, mathworks, natick, ma, usa). on the mobile application, for each test, the participants were required to rate their wellbeing "how do you feel today?" using a visual analogic scale (vas) graduated from to . all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint all results are given as mean ± sd. the kolmogorov-smirnov test was used to ensure normality of the data. measurements were evaluated with a two-way anova analysis for time effect (bsl, cfn and rcv) and group effect (wb-vs. wb+). the statistical power of the performed tests was set at p = . for significance and p = . for tendency. the tukey-kramer post-hoc was used when appropriate. pearson's coefficient correlations and significance were computed for lf, hf, hr and rmssd against vas, in the supine and the standing positions. statistical analyses were performed using matlab ® . ninety-five participants ( women, men) were included in this report (age ± years, height ± cm, weight ± kg). out of these participants, % (wb+, n = ; women, men; age ± years, height ± cm, weight ± kg) showed an improved well-being, estimated by the positive change (> ) in the - vas score between bsl and cfn. contradictory, most of the participants (wb-, n = ; women, men; age ± years, height ± cm, weight ± kg) did not improve or deteriorated this vas score during the cfn period. figure shows the negative impact of cfn in the wb-group, with a general alteration in parasympathetic activity in the supine position, as shown by the increased supine hr and decreased rmssd in cfn and rcv compared to bsl. there was a tendency for a decrease in rmssd between cfn and rcv. there was also a decrease in hf in rcv compared with bsl. in the standing position, there was a significant increase in hr in cfn and rcv compared to bsl, but there was no change on the other parameters. figure shows the positive impact of cfn in the wb+ group with a decrease in supine hr in cfn and rcv compared to bsl, an increase in supine rmssd in cfn and rcv compared to bsl and an increase in rcv compared to cfn. there was also an increase in lf and hf (and therefore total power) in rcv compared to bsl, and an increase in hf in rcv compared to cfn. there was no change in the standing position. there was an interaction time x group for hr, rmssd and hf (p < . , p < . and p < . , respectively) in the supine position. there was a significant interaction for hr only (p< . ) in the standing position. in the supine position, hr was significantly higher in the wb-group compared to the wb+ group in cfn and rcv. rmssd was significantly lower in the wb-group compared to the wb+ group in cfn and rcv. hf was significantly lower in the wb-group compared to the wb+ group in rcv. in the standing position hr was significantly higher in the wb-group compared to the wb+ group in cfn. there were significant correlations (p < . for all) between vas and hr ((r = - . ), rmssd (r = . ) and hf (r = . ) on the whole population in the supine position. the higher the vas score (i.e., subjective well-being), the higher the rmssd and hf and the lower the hr (figure ). when dividing the cfn period in two sub-periods of weeks, there were no differences in any of the parameter between cfn and cfn , suggesting a relative stability across cfn (data not shown). the present study aimed to investigate the hrv responses of participants exposed to the strict lockdown that occurred in france. we found that hrv changes from bsl to cfn and rcv were contradictory between two groups who did vs. did not deteriorate their subjective well-being over these periods. in % of the participants, parasympathetic-related hrv parameters (i.e., rmssd, hf) measured in the supine position were decreased concomitantly to a psychological alteration. contradictory, in % of the participants, both supine hrv and well-being were improved. standing hrv was not modified during cfn. the sudden lockdown that occurred implied drastic changes in the lifestyle of the participants, which is generally a stressor factor. these lifestyles and behaviors included a given level of physical exercise to maintain an adequate health status [ ] , and guarantee an active aging by reducing the risk associated diseases in older people [ ] . moreover, the psychological impact of lockdown included negative psychological effects, such as frustration, boredom, loss of social contacts, [ ] inadequate supplies, inadequate information, financial loss, infection fears, confusion, and anger [ , ] . the participants in the present study lived in france, where a survey of the french national public health institute (santé publique france) demonstrated a drastic decrease in physical activity and increased time spent in the seated position [ ] . in the present study, physical activity was not measured, but only subjective feeling of well-being, measured by a vas with a smartphone. this resulted in % of the participants who rated positively the lockdown period, since vas score improved by at least on the - scale. interestingly, this is in line with data from the french national public health institute, who reported that % of the french population declared an increase in physical activity. obviously, well-being can be determined by many causes and physical activity is one of them. in the present study, % of the participants reported an increased hr associated to decreased rmssd and increased lf: these changes could be the consequences of a cardiovascular detraining, with decreased parasympathetic influences on the heart. the loss of outdoor activities as walking is likely of clinical importance since lockdown accelerates vicious circles between isolation, lack of physical activity, psychological stress/anxiety and impaired immunity [ ] . meanwhile, the participants had more time to exercise, but they had to do it by themselves, based on their personal knowledge and material possibilities, with extremely limited external advises and totally absent group motivation. according to the french national public health institute, only % of the french population was able to increase the physical activity during the lockdown period. this likely resulted in only % of the participants in the present study able to maintain or improve their hrv parameters as shown on figure . in this group, the fact that increased rmssd, lf and hf (associated to decreased hr) were also observed post lockdown, is a marker of an effective and lasting improvement in cardiovascular fitness. the best performances occur when both the sympathetic and parasympathetic influences on the heart are high [ , ] hence increased lf and hf, associated to decreased resting hr. there was a moderate but significant correlation between vas and hrv, which was positive for hf and rmssd and negative for hr. therefore, the better the participants felt, the higher the parasympathetic influences and the lower the hr, and vice-versa the lower the hf and rmssd, the lower the feeling of well-being. these later results emphasize the usefulness of hrv as a global index of heath in the general population. in line with previous literature and all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . data from the national french public health institute, the more active people during the lockdown may have felt better [ , ] . the lockdown was applied suddenly, and it was therefore impossible to recruit participants a priori during the baseline period. by chance we had enough participants with sufficient hrv tests to conduct this study. overall, it is a unique opportunity to describe the hrv behavior in healthy people, extracted from a database of orthostatic tests. the main limitation is that we did not have any data on physical activity of these participants. we can only speculate that the hrv changes during cfn reflect at least partly the decrease or increase in physical activity. the lockdown in france was sudden and severe, drastically limiting the possibilities for physical activities. this likely resulted in a stressful situation and in a general cardiovascular detraining (i.e., decreased rmssd and hf and increased resting heart rate in supine position) in most of the population. however, in % of the participants the lockdown was positively appreciated, as shown by an improved well-being and parasympathetic activity. altogether, the present study confirms the usefulness of regular hrv testing in the general population for monitoring health status, as previously shown in elite athletes. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . figures figure : hr, rmssd, lf and hf before (bsl), during (cfn) and after (rcv) the strict lockdown period in france in participants who deteriorated or maintained their well-being over the cfn period. * p < . for difference with bsl. # p < . for difference with cfn. figure : hr, rmssd, lf and hf before (bsl), during (cfn) and after (rcv) the strict lockdown period in france in participants who improved their well-being over the cfn period. * p < . for difference with bsl. # p < . for difference with cfn. nb, sy, ls and gpm have no conflicts of interest, sources of funding, or financial ties to disclose and no current or past relationship with companies or manufacturers who could benefit from the results of the present study. nb and sy are employees of be.care sa. authors nb and sy were employed by the company becare sa. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august , . . https://doi.org/ . / . . . doi: medrxiv preprint metabolic impacts of confinement during the covid- pandemic due to modified diet and physical activity habits confinement : un impact certain sur l'activité physique, le temps passé assis et le temps passé devant un écran. press release exercise and the cardiovascular system: clinical science and cardiovascular outcomes physical activity and depression: type of exercise matters typology of "fatigue" by heart rate variability analysis in elite nordic-skiers the relationships between heart rate deceleration capacity and spectral indices of heart rate variability during different breathing frequencies heart rate variability indices as bio-markers of top-down self-regulatory mechanisms: a meta-analytic review power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control low-frequency power of heart rate variability is not a measure of cardiac sympathetic tone but may be a measure of modulation of cardiac autonomic outflows by baroreflexes assessment of autonomic function in humans by heart rate spectral analysis neural regulation of heart rate variability in endurance athletes and sedentary controls tapering for marathon and cardiac autonomic function effect of cold water immersion on -m sprint performance in well-trained swimmers monitoring training status with hr measures: do all roads lead to rome? associations of physical activity, fitness, and body composition with heart rate variabilitybased indicators of stress and recovery on workdays: a cross-sectional study minimal window duration for accurate hrv recording in athletes tracking morning fatigue status across in-season training weeks in elite soccer players sedentary behavior, exercise, and cardiovascular health an update on the role of cardiorespiratory fitness, structured exercise and lifestyle physical activity in preventing cardiovascular disease and health risk the psychological impact of quarantine and how to reduce it: rapid review of the evidence human needs in covid- isolation physical exercise as therapy to fight against the mental and physical consequences of covid- quarantine: special focus in older people indoor) isolation, stress and physical inactivity: vicious circles accelerated by covid- ? key: cord- -wk k igu authors: chandrasekaran, alamelu; manji, ryhana; joseph, ansamma; zhang, fan; ginocchio, christine c. title: broad reactivity of the luminex xtag respiratory virus panel (rvp) assay for the detection of human rhinoviruses date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: wk k igu nan human rhinoviruses (hrv) are a major cause of the common cold and a variety of both upper and lower respiratory tract infections including sinusitis, otitis media, bronchitis, primary pneumonia and have been associated with the worsening of asthma and chronic obstructive pulmonary disease. with the advent of new antivirals directed at the treatment of serious hrv infections it is becoming increasingly important to detect a broad range of hrv strains so that targeted antiviral therapy can be appropriated instituted. , the xtag respiratory virus panel (rvp, luminex molecular diagnostics, toronto, canada) is a us food and drug administration (us fda) cleared multiplex nucleic acid amplification test for the detection of respiratory viruses including adenovirus (adv), human metapneumovirus (hmpv), influenza a (influa with subtyping of seasonal h and seasonal h ), influenza b (influb), parainfluenza types (piv- ), (piv- ) and (piv- ), respiratory syncytial virus (rsv) a and b, and hrv. some enteroviral (ev) strains are also detected due to cross reactivity with hrv and therefore the assay does not distinguish between hrv and ev. , additional viruses detected by the full rvp (research use only in us) include piv- , coronaviruses (corona) nl , oc , hku- and e. a multicenter study demonstrated that rvp can detect culture isolates of hrv serotypes and and hrv strains of phylogenetic groups a, b, c, e and f from clinical samples. the aim of this study was to further assess the analytical reactivity of rvp for the detection of a wide variety of hrv serotypes and in clinical samples previously identified as hrv/ev positive by rvp. differentiation of hrv from ev in rvp hrv/ev positive samples was established using a laboratory developed hrv assay (ldthrv) based on a modified version of the method described by lu et al., and the nuclisens easyq enterovirus assay (eva, biomérieux, durham, nc). a l aliquot from cultures of hrv species a and b (n = , serotypes - , with the exception of serotypes and ), influa, influb, rsv a and b, and adv, and l aliquots from discard clinical samples (nasopharyngeal swabs in universal transport media [utm, copan, brescia, italy]) were added to . ml nuclisens lysis buffer tubes (biomérieux). nucleic acids (na) were extracted using the nuclisens easymag (biomérieux) and tested with ldthrv, eva and rvp according to the manufacturer's instructions. all hrv isolates were detected by rvp and ldthrv. eva was negative for / hrv isolates tested. the remaining hrv isolates ( serotypes , , , , , , , , , , , , ) gave positive eva results of varying intensity. the hrv isolates had culture titers ranging from a tcid of . × − to . × − and demonstrated varying levels of rvp reactivity with mfi values ranging from to . eva results were negative for isolates after testing ten-fold dilutions of the original culture. nucleic acids derived from clinical samples that contained hrv species c tested positive with rvp, ldthrv and negative with eva. discard clinical samples, previously determined by rvp to be only hrv/ev positive (+) (n = ), hrv/ev(+) with an additional virus (n = ; hmpv, piv- , piv- , rsv-b), hrv/ev negative (−) but other respiratory virus (+) (n = ; adv, corona nl , influa, influb, hmpv, piv- , piv- , piv- , piv- , rsv-a, rsv-b), gave the expected results. all samples positive for hrv/ev by rvp were detected by ldthrv and were negative by eva. all samples negative for hrv/ev by rvp were negative by ldthrv and eva. in conclusion, the xtag rvp can detect a broad range of hrv serotypes, including hrv a, b and c strains. although the assay cannot differentiate between hrv and ev, supplemental assays can be used to differentiate hrv and ev when clinically indicated. the detection of hrv will facilitate the use of appropriate targeted antiviral therapy. this study was funded in part by biota holdings, australia. ccg has received research funding from luminex and is a member of the luminex scientific advisory board. not required. the abcs of rhinoviruses, wheezing, and asthma study of the biological activity of novel synthetic compounds with antiviral properties against human rhinoviruses development of a respiratory virus panel test for the detection of twenty human respiratory viruses by use of multiplex pcr and a micro-bead based assay xtag tm rvp assay: analytical and clinical performance real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses development, technical performance, and clinical evaluation of a nuclisens basic kit application for the detection of enterovirus rna in cerebrospinal fluid ginocchio a,b, * a department of pathology and laboratory medicine, division of infectious disease diagnostics nucleic acid extracts from specimens previously characterized as positive for rhinovirus group c by molecular detection and sequencing, were kindly provided by dr. kirsten st. george at the laboratory of viral diseases, wadsworth center, new york state department of health, albany, ny. human rhinovirus strains were kindly provided by dr. simon tucker of biota holdings limited, notting hill, vic, australia. key: cord- -n jc jy authors: selvaggi, carla; pierangeli, alessandra; fabiani, marco; spano, lucia; nicolai, ambra; papoff, paola; moretti, corrado; midulla, fabio; antonelli, guido; scagnolari, carolina title: interferon lambda – expression in infants hospitalized for rsv or hrv associated bronchiolitis date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: n jc jy objectives: the airway expression of type iii interferons (ifns) was evaluated in infants hospitalized for respiratory syncytial virus (rsv) or rhinovirus (hrv) bronchiolitis. as an additional objective we sought to determine whether a different expression of ifn lambda – was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established ifn stimulated genes (isgs), such as mixovirus resistance a (mxa) and isg . methods: the analysis was undertaken in infants with rsv or hrv bronchiolitis. nasopharyngeal washes were collected for virological studies and molecular analysis of type iii ifn responses. results: rsv elicited higher levels of ifn lambda subtypes when compared with hrv. a similar expression of type iii ifn was found in rsva or rsvb infected infants and in those infected with hrva or hrvc viruses. results also indicate that ifn lambda and ifn lambda – levels were correlated with each other and with mxa and isg -mrnas. in addition, a positive correlation exists between the ifn lambda levels and the clinical score index during rsv infection. in particular, higher ifn lambda levels are associated to an increase of respiratory rate. conclusions: these findings show that differences in the ifn lambda – levels in infants with rsv or hrv infections are present and that the expression of ifn lambda correlates with the severity of rsv bronchiolitis. keywords ifn lambda; il- ; il- ; rsv; hrv; mxa; isg ; viral load; bronchiolitis summary objectives: the airway expression of type iii interferons (ifns) was evaluated in infants hospitalized for respiratory syncytial virus (rsv) or rhinovirus (hrv) bronchiolitis. as an additional objective we sought to determine whether a different expression of ifn lambda e was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established ifn stimulated genes (isgs), such as mixovirus resistance a (mxa) and isg . methods: the analysis was undertaken in infants with rsv or hrv bronchiolitis. nasopharyngeal washes were collected for virological studies and molecular analysis of type iii ifn responses. results: rsv elicited higher levels of ifn lambda subtypes when compared with hrv. a similar expression of type iii ifn was found in rsva or rsvb infected infants and in those infected with hrva or hrvc viruses. results also indicate that ifn lambda and ifn lambda e levels were correlated with each other and with mxa and isg -mrnas. in addition, a positive correlation exists between the ifn lambda levels and the clinical score index during rsv infection. in particular, higher ifn lambda levels are associated to an increase of respiratory rate. conclusions: these findings show that differences in the ifn lambda e levels in infants with rsv or hrv infections are present and that the expression of ifn lambda correlates with the severity of rsv bronchiolitis. ª the british infection association. published by elsevier ltd. all rights reserved. bronchiolitis is a disorder most commonly caused in infants by viral lower respiratory tract infections; it is characterized by acute inflammation, edema and necrosis of epithelial cells lining small airways, increased mucus production, and bronchospasm. the most common virus causing bronchiolitis is the respiratory syncytial virus (rsv). , other viruses identified as causing bronchiolitis are rhinovirus (hrv), human metapneumovirus, bocavirus, and parainfluenza. in particular, hrv has been recently shown to infect the lower airway as well and confirmed to be the second most frequent cause of bronchiolitis. it has been demonstrated that the combination of both host and viral factors profoundly influence the severity of viral associated bronchiolitis. e however, it is not yet clear whether the different subtypes of rsv (a and b) or hrv species (a, b, and c) cause different grades of bronchiolitis severity. , furthermore, the role of the innate immune response, in the pathogenesis of severe rsv or hrv disease is still to be defined in detail. e among the main players of antiviral innate immune response, the type i interferons (ifns), ifn alpha and beta, are considered cytokines crucial for anti-viral resistance and represent an early antiviral host defense mechanism against viral infections. in , a novel class of antiviral cytokines was discovered, characterized and classified as type iii ifns: ifn lambda /il- , ifn lambda /il- a, and ifn lambda /il- b. at the amino acid level ifn lambda and lambda are highly similar having % sequence identity while ifn lambda shares approximately % sequence identity with ifn lambda and lambda . the type iii ifns possess antiviral properties similar to those of type i ifns but appear to be expressed especially by epithelial cells and consequently exert host protection primarily at epithelial surfaces. e despite the fact that it is known that ifn lambda contributes to the control of viral infections in epithelial cells of respiratory tract e and that the presence of single nucleotide polymorphism around ifn lambda (il- b) can increase the risk of hospitalization for bronchiolitis at early age, the ifn lambda e expression in the respiratory tracts of hospitalized infants with rsv or hrv infections has never been addressed. hence, considering the importance of the ifn lambda in protecting the airway tract from virus infections, e we hypothesized that the heterogeneity of ifn lambda e levels could, at least in part, explain the broad clinical spectrum of rsv or hrv bronchiolitis. therefore, we evaluated whether there was a difference in the gene expression of ifn lambda e subtypes between infants with a clinical diagnosis of rsv associated acute bronchiolitis and those with hrv infection. the same analysis was also performed between rsv or hrv subtypes. in addition, to characterize the activation of type iii ifns in the airway tract of infants with rsv or hrv infections, we evaluated whether there was a coordinate activation between ifns lambda and that of mxa and ifn-stimulated gene (isg) , which are well known markers of type i and iii ifn antiviral activity. finally, to further characterize the above issues, we also assessed whether a correlation between ifn lambda e levels and demographic, virological and clinical parameters in rsv and hrv infected infants actually exist. a total of infants with single rsv or hrv infection were retrospectively selected from a total of infants admitted with a clinical diagnosis of acute bronchiolitis during three epidemic seasons ( e ) to the paediatric department of policlinico umberto i hospital. the study was approved by the ethics committees and informed consent was obtained from the infant's parents. bronchiolitis was diagnosed from the presence of a history of upper respiratory tract infection followed by the acute onset of respiratory distress with cough, tachypnea, retraction, and diffuse crackles on auscultation (wheezing alone was not considered sufficient cause for inclusion in the study). the exclusion criteria were underlying chronic disease (such as cystic fibrosis, chronic pulmonary disease, congenital heart disease, and immunodeficiency) and recurrent (more than one) wheezing episodes. , the severity of the illness was assessed clinically on the following four indications, each of which was assigned a score within the range e . in particular, on admission to hospital, the clinical severity was assigned to each infant, based on respiratory rate (< breaths/min z , e breaths/min z , > breaths/min z ) arterial oxygen saturation in room air (> % z , e % z , < % z ), presence of retractions (none z , present z , present þ nasal fare z ), and ability to feed (normal z , reduced z , endovenous z ). nasopharyngeal washings were collected in the first h after admission to the hospital from infants suffering from acute bronchiolitis, and an aliquot was tested for viruses as previously described. in particular nasopharyngeal washings were obtained with ml of sterile saline physiological solution injected into each nostril and collected with a syringe. all samples were delivered on ice within e h to the virology laboratory and on arrival, if needed, they were vortexed with beads to solve mucus. they were divided into two aliquots: one was treated for nucleic acid extraction and viral detection; the second was centrifuged at rpm for min, and each cell pellet was resuspended in ml of phenol and guanidine isothiocyanate reagent (trizol, gibco-brl, ny) and frozen at À c for gene expression analysis. a panel of reverse transcription-pcr (rt-pcr) or nested pcr assays, some in a multiplex format, were used for the detection of respiratory viruses, including rsv; influenza viruses a and b; coronaviruses oc , e, nl , and hku ; metapneumovirus; adenovirus; hrv; and parainfluenza virus types e , human bocavirus as previously reported. , the evaluation of the sensitivity of the rt-pcr or pcr tests for the respiratory viruses was made as described in our previously published papers. , in particular, the integrity of the extracted nucleic acid was tested by means of the amplification of the cellular gene beta actin. rsv or hrv-positive samples were typed as rsva-b or as hrv a-c respectively. the rsv fragment to be sequenced was obtained by re-extracting rna from rsv-positive samples and directly amplifying it using superscript one-step rt-pcr for long templates kit (life technologies, monza, italy) with two expressly designed forward primers, a-fseq -aat gat ttt cac ttt gaa- ; b-fseq -gat gat tac cat ttt gaa- , corresponding to g gene position e of rsv-a and to position e of the rsv-ba reference strains, respectively, and with one reverse primer targeting the fusion protein gene end. hrv-positive samples were retrospectively amplified with primers widely used for genotyping targeting bases of the untranslated region ( utr) central portion. the mrna copy content of ifn lambda e , mxa and isg was measured by a real-time exonuclease rt-pcr assay using the light cycler sequence detector (roche, monza, italy). briefly, the total cellular rna was extracted from the cells collected from nasopharyngeal washings as described, using phenol and guanidine isothiocyanate reagent (trizol, gibco-brl, ny), by following the manufacturer's instructions, and was retro-transcribed as previously specified. primer pairs and probes for ifn lambda [forward primer, -ggacgccttggaagagtcact- ; reverse primer, -agaagcctcaggtcccaattc- ; probe, fam-agttgcagctctcctgtcttccccg- 'tamra ], ifn lambda e [forward primer, -ctgccacatagcccagttca- ; reverse primer, -agaagcgactcttctaaggcatctt- ; pro be, fam-tctccacaggagctgcaggccttta- 'tamra ], mxa (forward primer, -ctgcctggcagaaaaacttac- ; reverse primer, -ctctgttattctctggtgagtctcctt- ; probe, fam catcacacatatctgtaaatctctgcccctgtt- 'tamra); isg [forward primer, -tgaagaagctctagc-caacatgtc- ; reverse primer, -gagctttatccaca-gagccttttc- ; probe: 'fam -tatgtctttcgatatg cagccaagttttaccg- tamra ] were added to the universal pcr master mix (roche) at and nm, respectively, in a final volume of ml. the coamplification of the beta-glucuronidase gene (forward primer, -tctgtcaagggcagtaacctg- ; reverse primer, -gcccacgactttgttttctg- ; probe, fam-tcaagttggaagtgcgtcttttggatgc- 'tamra) was used to normalize the amount of total rna present using the threshold cycle relative quantification [the e(delta) ct] method according to the supplier's guidelines. all the determinations were performed in duplicate. a taqman-based real-time pcr technique for rsv or hrv rna quantification was performed on all nasopharyngeal washing specimens with positive rt-pcr results for rsv or hrv respectively. briefly, viral rna was extracted from nasopharyngeal washings npw that were positive for rsv or hrv, using a qiaamp viral rna mini kit (qiagen, milan, italy). the rna was dissolved in rnase-free water and the rsv quantification was performed by taqman assay after generation of cdna using a high capacity cdna archive kit (applied biosystems, monza, italy). type-specific primers and probes for n gene of both rsv a and b or utr region of hrv a-c strains were added to the universal pcr master mix (roche) at and nm, respectively, in a final volume of ml. the standards for rsv or hrv were obtained respectively by cloning the bp of rsv n gene or bp of the utr hrv region into the pcr . plasmid using a topo ta cloning kit (invitrogen corporation, san diego, ca, usa). a linear distribution (r z . ) was obtained between and copies of rsv or hrv-dna. viral load values were log transformed for analysis and data was expressed as the log number of rsv or hrv copies per ml of nasopharyngeal washings. all the determinations were performed in duplicate. all measurements are expressed median (range) or frequency (percentage), unless otherwise indicated. the demographic and clinical characteristics of infants suffering from rsv or hrv associated bronchiolits were compared using the mannewhitney test. differences in the clinical score index values were analyzed using student's t test. differences between infants with rsv or hrv infections and between rsv (a and b) or hrv (a and c) strains, in terms of the level of ifn lambda e measured in cells from nasopharyngeal washings, were compared using the man-newhitney test. spearman's rho coefficient was calculated in order to assess the correlation between the level of ifn lambda and ifn lambda e and between ifn lambda e and isgs, demographic, clinical and rsv or hrv viral load. differences in the ifn lambda levels in rsv infected infants divided into groups on the basis of the respiratory rate were evaluated by using kruskalewallis test. the significance was fixed at the % level. analysis was performed with spss v. . for windows. one hundred and eighteen infants, admitted over a period of years to the paediatric department of policlinico umberto i university hospital with a diagnosis of single rsv or hrv associated bronchiolitis were included ( table ) . as far as virological characteristics are concerned, a total of ( %) infants carried a single rsv infection the clinical severity was assigned to each infant with the range e , based on respiratory rate (< breaths/min z , e breaths/ min z , > breaths/min z ), arterial oxygen saturation in room air (> % z , e % z , < % z ), presence of retractions (none z , present z , present þ nasal fare z ), and ability to feed (normal z , reduced z , endovenous z ). whereas ( %) had an hrv single infection. in particular % ( / ) of the rsv positive infants had a rsva infection and the remaining had a rsvb infection. among hrv infected infants, % ( / ) had an infection with hrva and % ( / ) had an hrvc infection. interestingly, no infants had an hrvb infection. when we analyzed the demographic and clinical parameters of infants with rsv or hrv infection, there were no significant differences between infants with rsv or hrv infection and between infants with different rsv (a vs b) or hrv (a vs c) strains (table ) . on the contrary the differences between infants with rsv or hrv infection were statistical significant when the clinical score index, and the percentage of infants with fever were analyzed (table ) . moreover, the number of eosinophils was lower in infants with rsv infection compared to those with hrv infection ( table ) . the airway transcription levels of ifn lambda and ifn lambda e were evaluated using real-time rt-pcr in cells from nasopharyngeal washings collected from infants with rsv infection (n z ) or hrv infections (n z ). the gene expression level of ifn lambda and ifn lambda e showed high variability between infants with rsv or hrv infection [(coefficient of variation > %), fig. as reported in fig. (panel a) the transcript levels of ifn lambda and ifn lambda e in infants suffering from rsv infection were higher than in those with hrv infection [ifn lambda (rsv vs hrv): p z . ; ifn lambda e (rsv vs hrv): p z . ]. furthermore, we found no differences between mrna levels of ifn lambda and those of the ifn lambda e in rsv or hrv infections (fig. , panel a) . no significant differences in transcript levels of type iii ifn were observed between infants with different rsv (a vs b) or hrv (a vs c) strains (fig. , panel bec). in order to characterize the activation of type iii ifn response during bronchiolitis, we also evaluated whether there was a coordinate activation of ifn lambda subtypes in the airway tract of infants suffering from bronchiolitis. results indicate that in the respiratory tract of infants with rsv or hrv the transcript levels of ifn lambda were significantly correlated with those of ifn lambda e (table ) . furthermore, considering that ifn lambda induces the expression of ifn-stimulated genes (isgs), we evaluated whether there was a correlation between the expression of ifn lambda subtypes and that of well established isgs, namely mxa and isg . results indicate that type iii ifn mrna levels were significantly correlated with the transcript expression of mxa and isg in infants with rsv or hrv bronchiolitis ( table ) . the relationship between patient data as independent variables and ifn lambda e mrna levels measured in nasopharyngeal washings in infants with rsv or hrv bronchiolitis was analyzed (table ) . we found a significant positive correlation between the transcript level of ifn lambda and the clinical score index in infants with rsv infection (r z . , p z . ) but not in those hrv infected (table ). in particular, ifn lambda seems to be associated to an increase in the respiratory rate during rsv infection. indeed, as showed in fig. , when rsv infected infants were divided into groups on the basis of the respiratory rate (< breaths/min, e breaths/ min, and > breaths/min), there was a significant difference between the groups (p z . ). specifically, infants with > respiratory breaths per minute showed higher gene expression of ifn lambda compared with infants with < or e respiratory breaths per minute. in contrast, we failed to detect any correlation between the ifn lambda e gene expression levels and age, weight, number of days of hospitalization, and several immunological and biochemical parameters (numbers of neutrophils, lymphocytes, eosinophils or platelets, and levels of glycemia, sodium, c-reactive protein and hemoglobin). in addition, no differences were detected in ifn lambda e gene expression for male and female and between infants with fever or without fever (data not shown). in an attempt to determine whether rsv or hrv load influences the gene expression of type iii ifn, levels of rsv-or hrv-rna were analyzed respect to the expression of ifn lambda subtypes. no significant correlations were observed between rsv or hrv load and the levels of ifn lambda e (table ). it has been proposed that ifn lambdas are likely one of the main ifn produced during innate responses to respiratory viruses in the airway tract, , , , in line with the observations that the expression of the ifn lambda receptor is limited primarily to epithelial surfaces including that of the lung. , however the in vivo role of these cytokines in the host innate immune response to rsv or hrv infection is yet to be defined. our study gave some significant new insights into this complex issue. in particular, we found that in the respiratory tract of infants infected with rsv or hrv, there is a coordinate expression of different subtypes of ifn lambda: such an expression parallel also with those of mxa and isg , well established antiviral proteins induced by type i and iii ifns. this data could suggest that the airway tract is responsive to type iii ifns and that rsv or hrv caused a coordinate induction of ifn lambda subtypes that in turn can regulate antiviral pathways. however since both isgs analyzed are also regulated by ifn type i which is known to be produced during viral infections and to share the same pathways with ifn lambda, it remains unclear to which extent ifn lambda might specifically contribute to antiviral immunity against rsv or hrv in infants suffering from bronchiolitis. in this study we also compared the activation of ifn lambda e in the respiratory tract of infants suffering from rsv or hrv associated bronchiolitis. results demonstrated that in cells collected from nasopharyngeal washings of rsv positive infants there are higher mrna levels of type iii ifns compared to those observed in infants with figure gene expression of ifn lambda e during rsv or hrv bronchiolitis. the levels of type iii ifns were evaluated by using rt-taqman based real time pcr assays in cells from nasopharyngeal washings collected from infants suffering from rsv hrv infection. this is particularly interesting considering that it is known that the rsv ns and ns proteins can suppress in vitro the induction of ifn lambda. however, whether such response reflects host reactions for counteracting the ns /ns viral interference on ifn lambda induction is actually unknown. in our previous studies, we also observed a higher transcript level of isgs as well as of most pattern recognition receptors in infants with rsv compared to those with hrv infections. , all these findings may suggest that rsv infection are generally associate to a more robust innate immune response compared to those caused by hrv. in agreement, garcia et al. reported that concentrations of several cytokines in nasal wash tend to be higher in children with rsv than in those with hrv. in contrast, jartii et al. found that children with hrv-associated wheezing episodes show increased concentrations of th and th cytokines compared with those with rsv. however they measured cytokines concentrations in serum rather than respiratory tract secretions during wheezing episodes. as far as hrv is concerned, there are no studies on the evaluation of ifn lambda expression during hrv bronchiolitis: however wheezing during the first hrv infections is considered a risk factor for subsequent asthma development. interestingly, a deficient ifn lambda response to hrv infection has been reported in childhood in asthmatic subjects irrespective of their atopic status and in atopic patients without asthma. furthermore, contoli et al. reported a deficient induction of ifn lambda by hrv in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of hrv-induced asthma exacerbation and virus load in experimentally infected human volunteers. in apparent contrast, the presence of higher ifn lambda levels were recently associated with worsening illness of hrv associated asthmatic exacerbations in children. undoubtedly, the complex correlation between cytokine responses and viral infections deserves more studies to be performed carefully monitoring the various components of innate immunity during the natural course of respiratory viral infections. in this study, we also found no differences in the expression of type iii ifns between infants infected with rsva or rsvb subtypes and between those infected with hrva or hrvc strains, suggesting that the specific strain of rsv or hrv would not affect diversely the rate of activation of antiviral response. as far as the influence of specific rsv or hrv strains on the clinical course of bronchiolitis is concerned, no significant differences in the clinical characteristics between rsv (a vs b) or hrv (a vs c) infected infants were also found. however it must be considered that the samples size of infants analyzed in this study when divided according to the specific rsv or hrv strain was too small. conflicting results have been published on rsv (a or b) and hrv (a-c) association with recurrent gravity of respiratory diseases. , , e in particular, many, but not all, of the published studies , e found that hrv-c is associated with more severe lower respiratory disease and with wheezing, compared with hrv-a. nonetheless, because of the small size of hrv-positive specimens within each individual diagnosis category, most studies suffered from low power and were unable to detect significant differences among specific diagnoses, as discussed in one of the largest study ever. moreover, few data are available on the comparison of the ability of rsv a or b to modulate innate immune responses and no studies were performed on this issue for hrv a-c. therefore we retain that no definite conclusions can be drawn on such issues. interestingly, this study also demonstrated that levels of ifn lambda seem to be associated with severity of respiratory disease in rsv but not in hrv infected infants. this observation is not unusual for rsv pathogenesis. in fact, several studies have described severe rsv disease in children who have high levels of inflammatory cytokines and chemokines produced by innate immunity in respiratory secretions. , whether or not ifn lambda contributes to augmentation of inflammation in our rsv positive infants is currently unknown. however, in addition to their antiviral effects, type iii ifns have been shown to play a critical role in regulating the adaptive immune response by acting directly on th / polarization and cytokine production. , in particular, ifn lambda has been shown to increase production of il- , il- , and il- , with no concomitant increase in il- or tnf, suggesting it may not directly engender local tissue destruction, but could contribute to the inflammatory process during bronchiolitis. a reciprocal control of ifn lambda and th -associated cytokines seems also to exist. indeed, it has been shown that there is a reciprocal regulation between ifn lambda and il- or il- which are well established th cytokines associated with increased rsv disease severity. in addition, although type iii ifns seems to be activated during in vivo rsv infection, we observed that enhancement was not related to viral loads. indirectly the lack of a direct effect of ifn lambdas on viral replication may reflect a more relevant role of the cross-talk between inflammatory citokines and type iii ifn subtypes in determining the bronchiolitis clinical course. intriguingly, there are also indications that ifn lambda signaling may be resistant to feedback mechanisms targeting ifn alpha allowing it to be a more effective and durable antiviral and immunomodulator cytokine, at least under some circumstances, that could cause a prolonged, diffuse inflammatory response in the airway tract of rsv infected infants. the presence of this strong inflammatory response is consistent with the observation of the presence of string respiratory rates in rsv positive infants with increased ifn lambda levels. on the other hand, the reduced expression of ifn lambda subtypes observed in the airway tract of hrv infected infants compared to those with rsv infection, may not be enough to activate an excessive inflammatory response affecting the clinical course of bronchiolitis. in agreement with this hypothesis, wang q et al. found that mda deficient mice with reduced ifn lambda productions show less hrv induced airway inflammation. a greater number of eosinophils was also observed in hrv infected infants than in those with rsv accordingly to our previous study. in this regards it has been recently demonstrated that eosinophils may contribute to antiviral immunity and play a beneficial role in limiting viral respiratory lung dysfunction. however, it is also believed that eosinophilia is one of the atopic features that may contribute to the higher risk to develop recurrent wheezing and asthma. furthermore, in this study in line with previous studies , , a greater clinical severity in rsv infected infants than in those infected with hrv has been observed. therefore it is conceivable that the presence of reduced airway ifn lambda response in hrv infected infants than in those with rsv infections could reflect the presence of a milder bronchiolititis clinical course caused by hrv compared to that associated to rsv. indeed, miller et al. reported that ifn lambda levels were higher in wheezing children infected with hrv compared with no-wheezing and increased with worsening symptoms. alternatively, our data could indicate that an impaired ifn lambda production during hrv infection is present, not only in asthmatic subjects, , but also in infants with bronchiolitis which can be associated with the inability to control early virus replication and to mount an adequate th / th immune response which in turn may impact on recurrent wheezing predisposition and an exacerbation pathogenesis. these results on the presence of a positive correlation between ifn lambda levels and the clinical score of bronchiolitis in rsv infected infants does not seem to be in agreement with those obtained previously on the evaluation of isgs levels in infants with bronchiolitis. this discrepancy, although unexpected, might be explained considering that several host and viral factors independently from ifn lambda can regulate, directly or indirectly, the isgs production. , in addition it has been shown that the signaling pathways which lead to the activation of ifn regulatory factor can induce transcription of ifnstimulated response elements without the involvement of ifns. e thus it remains plausible that two different players of the same biological system might exert an opposite, complementary or simply additive effect on the clinical outcome of rsv bronchiolitis. all these findings would be greatly strengthened by comparing the level of expression of mrnas encoding type iii ifns with the level of expression of other cytokines or antiviral responses (e.g. type i ifns) in the cells from nasopharyngeal washings derived from infants with rsv or hrv associated bronchiolitis. this analysis could allow us to deep understand the complex picture of the airway ifn lambda response as well as the intensity and the dynamic nature of the antiviral and inflammatory pathways associated to type iii ifn response during pediatric lower respiratory tract infections. unfortunately the collected material was just enough to perform the experiments shown in the present study and the above issues could not be addressed. another important analysis which should be, but for the above reason have not been made, is the separate analysis of ifn lambda and ifn lambda subtypes expression in order to characterize the distinct contribute of these subtypes in rsv or hrv bronchiolitis. further studies specifically aimed to address these important issues are needed. in conclusion, we have shown for the first time to our knowledge that ifn lambda e are expressed in infants suffering from bronchiolitis although their levels may be different on the basis of which virus, rsv or hrv, has been detected in the respiratory tract. in addition, we have demonstrated that ifn lambda can influence the severity of bronchiolitis caused by rsv, but not by hrv, suggesting that the rate of activation of type iii ifn response may act as a double-edged sword in some circumstance during pediatric respiratory viral infections. however, it must be underlined that several factors associated or not with the ifn system may influence the clinical severity of bronchiolitis. the latter issue is exemplified by our observations about the opposite effect exerted by isgs and ifn lambda (this study) on the clinical outcome of bronchiolitis. all these findings highlight the importance of studying the interplay between the pathways of ifn lambda subtypes and those of other inflammatory cytokines or chemokines in order to deeply understand the influence of type iii ifn response on the clinical course of respiratory diseases. substantial variability in community respiratory syncytial virus season timing viral bronchiolitis for the clinician respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants the role of rhinovirus in asthma exacerbations newly identified human rhinoviruses: molecular methods heat up the cold viruses current concepts of the pathogenesis of rsv bronchiolitis the relationship between rsv bronchiolitis and recurrent wheeze: the chicken and the egg innate immune response and bronchiolitis and preschool recurrent wheeze respiratory syncytial virus infection and immunity human rhinoviruses antiviral activity of the interferon a family: biological and pharmacological aspects of the treatment of chronic hepatitis c il- , il- and their class ii cytokine receptor il- r ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo an important role for type iii interferon (ifn-lambda/il- ) in tlr-induced antiviral activity interferon-lambda contributes to innate immunity of mice against influenza a virus but not against hepatotropic viruses respiratory virus induction of alpha-, betaand lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells type-iii interferon, not type-i, is the predominant interferon induced by respiratory viruses in nasal epithelial cells lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections evaluation of interleukin b single nucleotide polymorphisms in infants suffering from bronchiolitis genetic variation at the il gene locus is associated with severity of respiratory syncytial virus bronchiolitis detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in human bocavirus infection in hospitalized children in italy circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community assay for ' noncoding region analysis of all human rhinovirus prototype strains gene expression of nucleic acid-sensing pattern recognition receptors in children hospitalized for respiratory syncytial virus-associated acute bronchiolitis role of deficient type iii interferon-l production in asthma exacerbations evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus new molecular detection tools adapted to emerging rhinoviruses and enteroviruses simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus a and b by real-time pcr lambda interferon is the predominant interferon induced by influenza a virus infection in vivo il- a and il- mediate antiproliferative and antiviral signals in intestinal epithelial cells and murine cmv infection increases colonic il- a expression suppression of the induction of alpha, beta, and lambda interferons by the ns and ns proteins of human respiratory syncytial virus in human epithelial cells and macrophages upregulation of interferon-induced genes in infants with virus-associated acute bronchiolitis decreased innate immune cytokine responses correlate with disease severity in children with respiratory syncytial virus and human rhinovirus bronchiolitis systemic t-helper and t-regulatory cell type cytokine responses in rhinovirus vs. respiratory syncytial virus induced early wheezing: an observational study rhinovirus bronchiolitis and recurrent wheezing: -year follow-up deficient antiviral immune responses in childhood: distinct roles of atopy and asthma a mechanistic role for type iii ifn-l in asthma exacerbations mediated by human rhinoviruses human rhinovirus and human respiratory enterovirus (ev and ev ) infections in hospitalized patients in italy human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children molecular epidemiology and genetic diversity of human rhinovirus affecting hospitalized children in rome human rhinovirus c: age, season, and lower respiratory illness over the past decades similar cytokine profiles in response to infection with respiratory syncytial virus type a and type b in the upper respiratory tract in infants elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease chemokines and inflammation in the nasal passages of infants with respiratory syncytial virus bronchiolitis modulation of the human cytokine response by interferon lambda- (ifn-lambda /il- ) interferon lambda- (ifn-lambda /il- ) induces elr(-) cxc chemokine mrna in human peripheral blood mononuclear cells, in an ifn-gammaindependent manner ifn-lambda (il- ) inhibits gata expression and suppresses th responses in human naive and memory t cells usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response mda and tlr initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness incidence and predisposing factors for severe disease in previously healthy term infants experiencing their first episode of bronchiolitis eosinophils contribute to innate antiviral immunity and promote clearance of respiratory syncytial virus impaired innate interferon induction in severe therapy resistant atopic asthmatic children respiratory syncytial virus nonstructural proteins ns and ns mediate inhibition of stat expression and alpha/beta interferon responsiveness transcriptional profiling of interferon regulatory factor target genes: direct involvement in the regulation of interferon-stimulated genes distinct and essential roles of transcription factors irf- and irf- in response to viruses for ifn-alpha/beta gene induction multiple signaling pathways leading to the activation of interferon regulatory factor respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology this work was supported by a grant from "sapienza" university of rome to carolina scagnolari ("ricerche universitarie", anno , prot c a x hp). key: cord- - v asfa authors: asner, sandra; peci, adriana; marchand‐austin, alex; winter, anne‐luise; olsha, romy; kristjanson, erik; low, donald e.; gubbay, jonathan b. title: respiratory viral infections in institutions from late stage of the first and second waves of pandemic influenza a (h n ) , ontario, canada date: - - journal: influenza other respir viruses doi: . /j. - . . .x sha: doc_id: cord_uid: v asfa please cite this paper as: asner et al. ( ) respiratory viral infections in institutions from late stage of the first and second waves of pandemic a (h n ) , ontario, canada. influenza and other respiratory viruses ( ), e –e . we report the impact of respiratory viruses on various outbreak settings by using surveillance data from the late first and second wave periods of the pandemic. a total of / ( · %) outbreaks tested positive for at least one respiratory virus by multiplex pcr. we detected a(h n )pdm in · % of all reported outbreaks of which · % were reported by camps, schools, and day cares (csds) and · % by long‐term care facilities (lcfts), whereas enterovirus/human rhinovirus (ent/hrv) accounted for % outbreaks of which · % were reported by long‐term care facilities (lctfs). ent/hrv was frequently identified in ltcf outbreaks involving elderly residents, whereas in csds, a(h n )pdm was primarily detected. respiratory outbreaks are common in healthcare and community institutions such as long-term care facilities (ltcfs) and schools. , the most commonly identified viruses have been influenza and respiratory syncytial virus (rsv). , human rhinovirus (hrv) has more recently been identified as a major viral pathogen in ltcf outbreaks. recent data reported by public health ontario (pho) indicated that pandemic influenza a (h n ) [a(h n )pdm ] was a rare cause of ltcf respiratory outbreaks during the first period of wave i (april -june , ) of the pandemic. we used surveillance data from the late stage of the first wave and the duration of the second wave periods (june -november , ) to ascertain the impact of a(h n )pdm and other respiratory viruses on different outbreak settings such as ltcfs and schools. for the purpose of this study, we considered the period from april to august , , as wave i and the period from september to november , , as wave ii, although wave ii activity continued until january . we investigated all respiratory outbreaks in ltcfs and camps, schools, day cares (csds) tested at pho laboratories from june through november , , in ontario, canada. a confirmed respiratory infection outbreak in a ltcf, as defined by ontario's ministry of health (moh), requires two cases of acute respiratory illness within hours of which at least one has to be laboratory-confirmed, or three cases of acute respiratory illness occurring within hours in a geographic area, none of which are laboratory-confirmed. ]. an alternate multiplex naat kit (seeplex rv; seegene usa, rockville, md, usa) was used in conjunction with the luminex assay during periods of higher demand. an in-house assay specific for a(h n )pdm was also performed. a total of respiratory outbreaks in csds, ltcfs, and other facilities (hospitals, correctional facilities, and unknown) were reported to and tested at pho. molecular testing was performed on samples from different outbreak settings. the number of samples submitted per outbreak is shown in figure . of the average of four samples (range - ) tested per outbreak, the median number of positive samples per outbreak was (range - ). outbreak samples mostly originated from ltcfs ( ae %) and csds ( ae %). hospitals and correctional facilities comprised ( ae %) and ( ae %) of remaining outbreaks, respectively. facility type was unknown for ae % outbreaks tested. mean and median ages of all persons tested were ae and years, respectively (range - years). mean and median ages of ltcf outbreak cases were ae and years, respectively; csds had a much younger population with mean and median ages of ae and years, respectively. at least one viral agent was identified in ( ae %) of the outbreaks tested. of these, ( ae %) outbreaks had only one sample with a virus identified, and the remaining ( ae %) had the same virus identified in two or more samples (range - ). ent ⁄ hrv and a(h n )pdm were the most common viruses detected in ( %) and ( ae %) outbreaks, respectively. of the outbreaks in which a(h n )pdm was detected, ( ae %) occurred in csds, ( ae %) in ltcfs, and ( ae %) in other facilities. of the outbreaks in which ent ⁄ hrv was identified, ( ae %) occurred in ltcfs, ( %) in csds, and ( %) in other facilities ( figure ). both viruses were identified throughout the entire study period, reaching a peak during october of wave ii (figure ). two hundred and forty outbreaks ( ae %) were caused by a single virus, the most common being ent ⁄ hrv ( %) or a(h n )pdm ( ae %). one hundred and ninety of these single virus outbreaks occurred in ltcfs and in csds. more than one etiological agent was identified in ( %) of the outbreaks, of which ( %) had two or more viruses detected within the same sample (coinfection). the most common coinfection was a(h n )pdm ⁄ ent ⁄ hrv, detected in ( %) of the outbreaks with coinfections. in the three outbreaks where two or more samples had viral coinfection (range - samples with coinfection), the same two viruses were found in all coinfection samples. there was an increased likelihood of identifying multiple viruses if or more samples were tested (p < ae ; figure ). nineteen outbreaks with multiple viruses detected were reported in ltcfs and in csds. ent ⁄ hrv and a(h n )pdm were the most common co-circulating viruses reported in all outbreaks with multiple viruses detected, found in of the multiple virus outbreaks reported by ltcfs and of the multiple virus outbreaks reported by csds (table ) . was detected in the majority of respiratory outbreaks in ltcfs, where the vast majority of residents were elderly. recently published reports - have already highlighted these findings. however, this study provides a larger sample size and is unique in that both institutional (predominantly elderly) and community outbreak (predominantly children) settings are described. we also found an increased likelihood of detecting viruses causing outbreaks as the number of specimens tested increased. viruses causing coinfections may be co-transmitted between patients as part of the same outbreak, rather than circulating independently of each other. additional bacterial analysis would be required to conclude that hrv is the sole etiology of individual outbreaks. in contrast, the pandemic virus was detected in almost all csd outbreaks, which mostly involved children and younger adults as recently highlighted in the literature. , possible reasons for a lower prevalence of a(h n )pdm in ltcfs outbreaks include cross-protective antibodies from previous exposure to influenza a (h n ) strains among elderly or minimal exposure to individuals more likely to be infected by the pandemic strain, such as children. , from june to november , , we found that ent ⁄ hrv was frequently identified in ltcf outbreaks involving elderly residents, whereas in outbreak settings involving children and younger adults, a(h n )pdm was primarily detected. younger children were not well represented in the csd group, which had a median age of ae years. this likely reflects the age distribution of children that attend sleep away summer camps in ontario. limitations of our study include its observational design, which limits establishment of causality and also limits our ability to exclude the effect of measured or unmeasured confounders in our analysis. a distinction between upper and lower respiratory tract infections, the role of asymptomatic shedding, and comparison of severity of outbreaks could not be performed as clinical data were missing. in ontario, the local medical officer of health or designate determines whether an institutional outbreak meets the provincial case definition. pho laboratories do not receive sufficient outbreak information to make this determination. in this context, this study highlights the importance of submitting more than one sample to properly investigate an outbreak. other bacterial agents like mycoplasma pneumoniae or chlamydophila pneumonia could not be ruled out as the cause of outbreaks as samples were not routinely tested for them. a distinction could not be made between staff and resident cases in this study as staff information was not systematically reported. this highlights a current deficiency in the investigation and reporting of staff illness in ltcfs. an increased rate of ent ⁄ hrv outbreaks may have been observed during the pandemic because of increased vigilance by individuals to report symptoms to their healthcare providers, who in turn had a lower threshold to report outbreaks to the public health units who coordinate laboratory investigations. therefore, we might have seen increased specimen collection and testing that may have influenced our findings. identification of specific non-influenza organisms associated with outbreaks can assist with outbreak management as the period of patient isolation and when to declare an outbreak over are dependent on the incubation period and period of communicability, which varies by organism. documentation of hrv as a cause of ltcf outbreaks is important, as recent studies suggest that hrv outbreaks can cause severe and fatal disease in ltcfs, especially among the elderly. a study comparing viral outbreaks in different community ⁄ facility settings with prospective gathering of detailed clinical and epidemiological information, supported by comprehensive microbiological analysis, will help further understand the role of different respiratory viruses as etiologic agents. rhinovirus outbreaks in long-term care facilities influenza and respiratory syncytial virus in the elderly respiratory infection in institutions during early stages of pandemic (h n ) ontario agency for health protection and promotion (oahpp) appendix b: provincial case definitions for reportable diseases analytical and clinical validation of novel real-time reverse transcriptase-polymerase chain reaction assays for the clinical detection of swine-origin h n influenza viruses a rhinovirus outbreak among residents of a long-term care facility uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold severe human rhinovirus outbreak associated with fatalities in a long-term care facility in ontario incidence of pandemic influenza a h n infection in england: a cross-sectional serological study estimated epidemiologic parameters and morbidity associated with pandemic h n influenza sandra asner, adriana peci, alex marchand-austin, anne-luise winter, romy olsha, and erik kristjanson have no conflicts of interest to declare.donald e. low participated in advisory board committee meetings for glaxosmithkline inc. and hoffman-la roche. he has also received research funding from both companies. jonathan b. gubbay has received a research grant from glaxosmithkline inc. to work on resistance to neuraminidase inhibitors. in june , oahpp received a research grant from glaxosmithkline to study phenotypic resistance in influenza virus. key: cord- -ava u y authors: rady, hanaa i.; kholy, amani el title: prevalence of human rhinovirus infection in young children with acute wheezing() date: - - journal: gaz egypt paediatr assoc doi: . /j.epag. . . sha: doc_id: cord_uid: ava u y introduction: recurrent wheezing is one of the leading causes of chronic illness in childhood. we aimed to evaluate the prevalence of human rhinovirus (hrv) infection in the acute attack of wheezy chest which began after a respiratory illness. methodology: the study was conducted on children aged months to years presenting to the emergency department with an acute wheezy episode either for the first time or recurrent wheeze defined as > reports of wheezing in the first years of life. all subjects were subjected to a complete history and clinical examination. chest x-ray was done to all subjects. nasopharyngeal and oropharyngeal swabs were obtained from all subjects and the presence of hrv was determined by pcr examination. results: by pcr method, patients ( . %) were positive for viral infection. due to viral co-infection, . % ( cases) were +ve for respiratory syncytial virus followed by hrv . % ( cases). conclusion: hrv was the second common viral infection in children with wheezes. its prevalence was more in winter with higher incidence of recurrence. compared to the other respiratory viruses, it had the higher mortality . %. asthma is a heterogeneous and multi-factorial disease that manifests as episodes of coughing, wheezing, and shortness of breath mainly at night. the major pathophysiology of asthma is bronchial inflammation with airway hyper-responsiveness, which results in reversible airway obstruction. among the various factors that have been involved in the pathogenesis of asthma, viral infections are the most prominent. viral infection affects wheezing and asthma in children and adults of all ages. wheezing illnesses are usually viral in origin, and children with more severe wheezing episodes are more likely to develop asthma later on in their life. human rhinoviruses (hrv) are not only the main pathogens responsible for the common cold, but are also now recognized to have a major impact on asthma pathogenesis. children who experience repeated rhinovirus-induced wheezing episodes in infancy have a significantly increased risk of developing asthma, even when compared to children who experience wheezing induced by respiratory syncytial virus (rsv). the aim of this study was to determine the prevalence of human rhinovirus as a cause of acute wheezing in egyptian children after an acute respiratory illness. a prospective study including children aged months to years presenting to the emergency department (ed) of cairo university children hospitals, with an acute wheezy episode (signs of respiratory distress and expiratory wheezes on auscultation and/or hyperinflation of the chest on chest radiograph) either for the first time or recurrent wheeze defined as > reports of wheezing in the first years of life. we excluded children with underlying cardiac or chronic pulmonary disease (other than asthma), the presence of stridor or egyptian pediatric association gazette j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e p a g daily treatment with oral corticosteroids for > days prior to presentation. all included cases had complete physical examination including grades of respiratory distress chest x-ray and laboratory investigations in the form of oxygen saturation, blood gases, and complete blood count. a nasopharyngeal and oropharyngeal swabs were taken and reverse transcription pcr was used to screen the samples for hrv. oropharyngeal swabbing: a dry sterile tip flocked with nylon fiber swab applicator was used to swab both the tonsils and the posterior pharynx. nasopharyngeal swabbing: a flexible sterile nylon fiber swab applicator was inserted into the nostril and back to the nasopharynx. it was then slowly withdrawn with a rotating motion. sample processing: the swabs were placed in a ml centrifuge tube labeled with the patient unique id and containing ml viral transport media (vtm: consisting of a sterile solution of bovine albumin fraction v, hepes buffer, penicillin, and streptomycin in hank's balanced salt solution). the received swabs inside the ml tube were agitated vigorously for s using a vortex mixer to free cells from the swab tip, and then both swabs were removed from the tube and discarded using a forceps. the sample was kept in a À c deep freezer until processed. nucleic acid extraction: automated extraction was performed using the qiacube machine with qiaamp Ò viral mini kit cat# , (qiagen) using the manual lysis protocol which consists of purification from manually lysed cell-free body fluids. multiplex real-time pcr was performed using the anyplex tm ii rv detection cat# rv g y from seegene inc, compatible with cfx tm real-time pcr bio-rad, the interpretation was done using the see gene viewer program. management of patients was followed depending on their condition whether received ambulatory therapy, required hospital admission or required intensive care unit admission. we recorded the length of hospital stay and patient's outcome: discharged, transferred or died. the study was explained for each parent before inclusion and an informed written consent was obtained from parents before enrollment. statistical package for social science (spss) version . was used for analysis of data. data were summarized as mean, sd and percentages. non parametric (mann-whitney u) test was used for analysis of quantitative data, as data were not symmetrically distributed. while chi square test was used for detection of risk factor for rhinovirus infection. p value was considered significant if < . . we studied children that presented to children hospital of cairo university ed with acute wheezy chest. the patients' condition varies from requiring nebulizer at the ed, hospital admission and oxygen supplementation to pediatric intensive care admission for infusion therapies or mechanical ventilation. the studied patients were male and female aged months to years. by pcr method, patients ( . %) were positive for viral infection and ( . %) patients were negative. from the viral infected patients patients ( . %) had single infection, while patients ( . %) had co-infection with more than one virus. rsv affected . % ( cases), followed by hrv . % ( cases) ( table ) . all the demographic and clinical data of cases were demonstrated in table . the median age of patients who were positive for rhinovirus was younger than rhinovirus negative patients, and this was statistically significant. regarding clinical data cough, grunting, fever and vomiting were more prevalent among hrv +ve patients p = . ; . ; . and . ; respectively, while cyanosis and diarrhea were more prevalent among hrv -ve patients p = . and . ; respectively. patients with history of recurrent wheezes ( . %) were more common than patients with first-time wheeze ( . %), p = . as shown in table . recurrent attacks of wheezes were more common among hrv +ve cases ( . %) compared to other viruses ( . %), a difference which was statistically significant p = . . there was a statistically significant difference between the frequency of rhinovirus positive and negative patients throughout the studied period. rhinovirus was most frequently detected throughout the winter months from december to february, as shown in fig. . table comparing the fate between patients positive for rhinovirus and negative for rhinovirus among virus positive patients: there was no significant difference regarding the illness severity between patients infected with hrv compared to other viruses: all patients who tested positive for rhinovirus infection by pcr were admitted to the pediatric intensive care unit (picu) with . % needing mechanical ventilation while . % of patients who tested negative for rhinovirus by pcr required icu admission with % needing mechanical ventilation, p = . . as regards outcome there was statistically significant difference in outcome between rhinovirus positive and negative cases. mortality rate was higher among hrv positive cases ( . %) vs ( . %), p = . . among children that presented to the ed of cairo university children hospital with wheezes, atopic wheezes were only in ( . %) while the majority was viral induced ( . %). the predominance of hrv infection among younger age was in accordance with guittet and colleagues, who studied children, median age was . ( . - . ) months, and where more than half, ( . %) were under months. jartti and colleagues, studied children hospitalized with acute expiratory wheezing and the presence of rhinovirus, rsv, coronavirus, metapneumovirus and enterovirus rnas were detected in the nasal secretions. in our study, we had almost the same viruses isolated from wheezy infants and children but rsv was most common, followed by hrv then adenovirus. another study done on hospitalized children admitted with acute expiratory wheezes, viruses were detected in the nasopharyngeal aspirates by pcr. a viral pathogen was identified in ( %). rsv was the most frequently detected ( %), followed by rhinovirus ( %). in our study, hrv +ve cases represented with recurrent attacks of wheezes rather than single attack. hrvs have been recognized as an extremely common cause of recurrent wheezing in early childhood by message and his colleagues, . hrv infection was associated with a -fold risk of developing asthma. among rhinovirus positive patients, co-infection with more than one virus was ( . %, cases) than infection with single virus ( . %, cases). fujitsuks and his colleagues , had co-infection detected in ( . %) patients while, smuts and his colleagues , it was %. , interestingly, our study comes in agreement with a study done by feng and his colleagues, which was performed on nasopharyngeal swabs obtained from children hospitalized for acute lower respiratory tract infection. approximately % were confirmed as hrv-positive cases with % confirmed as rsvpositive cases. this study shows also that hrv infection occurs sporadically throughout the year with an hrv-positive rate higher in winter and autumn. all our hrv +ve cases were admitted to picu and this could be explained by the presence of bacterial super-infection. this comes in agreement with a study done by guittet and colleagues . the results showed that . % of patients with rhinovirus showed pneumonia and % of them showed abnormal chest x-ray in the form of increased bronchovascular markings, consolidation, pneumothorax and pleural effusion. conclusion hrv was the second common viral infection in children with wheezes. its prevalence was more in winter with higher incidence of recurrence. compared to the other respiratory viruses, it had the higher mortality . %. no conflict of interest. no funding from any source. variants of dennd b associated with asthma in children gina guidelines on asthma and beyond rhinoviruses, allergic inflammation, and asthma role of rhinovirus infections in asthma rhinovirus and acute respiratory infections in hospitalized children. retrospective study persistance of rhinovirus and enteroviruses rna after acute respiratory illness in children role of emerging respiratory viruses in children with severe acute wheezing rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production the role of rhinovirus infections in the development of early childhood asthma a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness human rhinovirus infection in young african children with acute wheezing detection and clinical features of human rhinovirus in hospitalized children with acute respiratory tract infection in eastern areas of guangdong province i would like to thank all the icu team, the laboratory team and all my patients for their cooperation and sincerity during the study period. key: cord- - lb kho authors: nan title: oliv sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: lb kho nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- -kgb r authors: ahn, jong gyun; kim, dong soo; kim, ki hwan title: clinical characteristics and cytokine profiles of children with acute lower respiratory tract infections caused by human rhinovirus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: kgb r the clinical profile of human rhinovirus (hrv) with regard to lower respiratory infections remains unclear. we analyzed the clinical features and cytokine responses of hrv isolates in children with respiratory infections. quantitative analysis and genotyping of the hrv-positive samples from nasopharyngeal aspirates (npas) were performed using vp /vp sequencing. to compare t-helper (th ) type (ifn-γ, tnf-α) and th type (il- , il- ) cytokine responses between hrv-a, b and c, the levels of the four cytokines were measured. the hrv-positive children had shorter fever duration (p = . ), and higher frequencies of chest retraction (p = . ) and wheezing (p = . ) than did the hrv-negative group. hrv-a was identified in cases ( . %), hrv-b in ( . %), and hrv-c in ( . %). there were no significant differences in the clinical data or npa cytokines levels between patients with hrv-a and hrv-c infections. hrv is an important pathogen of the lower respiratory tract in young children. hrv-a and hrv-c are the dominant species that cause respiratory difficulty in young children. human rhinovirus (hrv) is the most common viral respiratory agent in humans. although it is the predominant cause of the common cold, hrv was recently found to be associated with an extensive range of more severe respiratory illnesses. this virus has been implicated in pneumonia, bronchiolitis, and exacerbation of asthma and chronic obstructive pulmonary disease [ ] [ ] [ ] [ ] . hrv is a small, non-enveloped single-stranded rna virus that belongs to the enterovirus genus and picornaviridae family. it is currently classified into three species on the basis of gene sequencing analysis. these include hrv-a, hrv-b, and the recently discovered hrv-c [ , ] . there are > distinct serotypes of hrv species according to their surface proteins. this biological diversity makes it difficult to develop vaccines or antivirals against hrv. understanding the epidemiological, clinical, and immunological features of hrv will help to prevent and treat hrv respiratory diseases. however, these characteristics have yet to be completely assessed. therefore, in this study, we investigated the epidemiological, clinical, and virological characteristics of hrv infections in children with acute lower respiratory tract infections. we also compared the t-helper (th )-and th -related cytokine profiles of hrv infection in nasopharyngeal aspirates (npas) to assess the immune responses of the disease. a total of nasopharyngeal aspirates (npas) were collected from children < years old hospitalized with acute lower respiratory tract infections at severance children's hospital in seoul, korea between march and january . as part of the diagnosis, pneumonia, croup, bronchiolitis, and asthma were included. these diseases were diagnosed through physical examination and chest x-rays. patients were excluded if they were immunocompromised due to hiv infection, malignancy, congenital or acquired immunodeficiency, solid organ transplantation or use of immunosuppressive drugs such as long-term systemic corticosteroids or radiation therapy. npas were obtained by suction using a fine, flexible plastic catheter and syringe. after collection, npas were immediately transported at ˚c to the laboratory, where they were stored at - ˚c until use. patient demographic and clinical data including medical history, detailed signs and symptoms, physical examination, and laboratory and radiology results were collected from a retrospective review of the medical records and interviews upon admission. disease severity was assessed according to a respiratory symptom scoring system based on previously published studies [ , ] . nucleic acid from each npa was extracted using a qiaamp viral mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. a one-step multiplex pcr/rt-pcr kit (solgent, daejeon, korea) was used to detect respiratory viruses [ ] . these included hrv, rsv, human bocavirus (hbov), influenza a virus (iav), influenza b virus (ibv), human metapneumovirus (hmpv), adenovirus (adv), human coronaviruses (hcov) e, oc , and parainfluenza viruses (piv) - . amplification was performed according to the manufacturer's protocol. the pcr products were run on a % agarose gel, stained with ethidium bromide, and visualized with uv light. hrv-positive specimens by multiplex pcr/rt-pcr were subsequently submitted to amplification reaction with the real-time rt-pcr assay using the powerchek™ rhinovirus real-time pcr kit (kogenebiotech, seoul, korea) and abi- fast real-time pcr system (applied biosystems, foster city, ca). rt-pcr was performed according to the manufacturer's instructions under the following conditions: initial denaturation at ˚c for min and ˚c for min, followed by cycles at ˚c for s and ˚c for min. ninety-four of the hrv-positive pcr samples were sequenced by solgent co. (daejeon, korea). the vp /vp sequences were aligned by clustral w. phylogenetic analysis was conducted using mega . software. the nucleotide and deduced amino acid sequences of the vp /vp regions were compared with reference sequences of hrv-a, hrv-b and hrv-c strains previously published in the genbank using blast (basic local alignment search tool -http://blast.ncbi.nlm.nih.gov/). we measured the concentration of the th (ifn-γ, tnf-α) and th (il- , il- ) cytokines in npa from hrv-positive patients for which genotyping using vp /vp sequencing was undertaken. the concentrations of the four cytokines in npa was measured using commercially available enzyme-linked immunosorbent assay kits (ebioscience, san diego, ca, usa) according to the manufacturer's instructions. statistical analysis was performed using spss (version . , chicago, il, usa). demographic, clinical, and laboratory parameters were compared between hrv-positive and -negative children and also among hrv species. pearson χ or fisher's exact test was applied for categorical variables. the student's t-test or non-parametric mann-whitney u test, anova tests or kruskal-wallis test were used for continuous variables, where applicable. p values < . were considered statistically significant. the study protocol was reviewed and approved by the yonsei university health system institutional review board, seoul, korea ( - - ). the study was conducted in accordance with good clinical practices (national regulations and ich e ) and the principles of the helsinki declaration. written informed consent was obtained from the parents or legal guardians of the patients prior to sample collection following a detailed explanation of schedules and contents of the study. the demographic, clinical and laboratory characteristics of all enrolled patients are shown in table . hrv was detected in ( . %) of npas. among the hrv-positive samples, ( . %) were co-detected with other respiratory viruses. hrv-infected children were significantly younger and had a shorter fever duration than the hrv-negative group. in contrast, the frequency of chest retraction and wheezing were significantly higher in the hrv-positive group than in the hrv-negative group. the white blood cell (wbc), hemoglobin (hb) and platelet (plt) counts were within normal range in both groups, although there were significant differences between groups. there were no other significant differences between the laboratory results of hrv-positive and -negative patients. between the hrv mono-and coinfection groups, there were no significant differences in clinical or laboratory data. the median viral load in hrv-positive patients was . × (interquartile range . × - . × ) rna copies/ml npa. however, there was no relationship between the hrv viral load and either clinical symptoms or laboratory data. all relevant data, inclusive of particular epidemiologic and clinical data as well as cytokine profiles, are included in a supplemental file (s table) . the viral protein vp /vp coding region was sequenced in of hrv-positive samples. phylogenetic analysis demonstrated that there were ( . %), ( . %) and ( . %) cases of hrv-a, -b and -c, respectively. hrv infection occurred throughout the year during the study period, although the peak prevalence of hrv-c was observed in autumn (fig ) . demographic, clinical, and laboratory findings between rhinovirus genotypes are shown in table . children with hrv-b were significantly older than those infected with hrv-c. clinical manifestations were not associated with any specific rhinovirus. the only exception was that none of the children with hrv-b infections had chest retraction, although the number of infections caused by this virus was low. there were no significant laboratory differences between hrv-a, b and c, except with regard to the wbc count, and percentage of neutrophils and lymphocytes. regardless, these values were all within the normal range in all three groups. the concentrations of ifn-γ, il- , il- , and tnf-α in npa of hrv-a-, b-and c-positive patients are shown in table . there were no significant differences in the npa cytokine levels between the three groups. this study demonstrates that hrv is an important cause of lower respiratory infection in young children that is associated with symptoms of respiratory distress, such as chest retraction and wheezing. our findings corroborate several recent studies reporting that hrv, which had previously only been known to cause upper respiratory infection, could play an important role in lower respiratory infection [ , [ ] [ ] [ ] . in our study, the majority of hrv genotypes were hrv-a ( . %) and hrv-c ( . %). the results of this study correspond with those of earlier studies, which found that hrv-a and -c were the predominant species, although the detection order depends on the geographic region [ , [ ] [ ] [ ] [ ] [ ] [ ] . previous studies have reported conflicting results with regard to disease severity according to hrv species. most of the earlier studies reported that hrv-c was associated with more severe respiratory disease in children [ , , , , ] . others found no clinical differences between the three hrv species [ , ] . further studies showed that both hrv-a and hrv-c were associated with acute respiratory infection hospital admissions, serious disease outcomes or wheezing episodes [ , , , ] . in our study, there were no significant differences in the clinical features, laboratory data or npa cytokine levels between hrv-a and -c infections. hrv-b seems somewhat different from hrv-a and -c. in accordance with previous studies [ , [ ] [ ] [ ] , we found that hrv-b was the least frequently detected hrv species, and chest retraction infections caused by this virus was not observed. there are two hypotheses to explain this. it is possible that hrv-b causes a mild form of disease that does not warrant hospitalization, accounting for its lower detection rate in hospital-based studies compared to those of the other hrv species. another possibility is that the genetic and biological fitness of hrv-b leads to its low frequency. one recent article reported that, even in healthy young children, hrv-b was the least frequently detected species of the three [ ] . this finding supports the hypothesis that the frequency and virulence of hrv-b may be related to its genetic fitness, rather than selection bias. there have been discrepant results regarding the correlation between the npa hrv viral load and disease severity. most previous studies reported that a high hrv viral load was correlated to clinical severity or wheezing [ ] [ ] [ ] . however, other reports show no correlation between the viral load and severity of the lower respiratory infection [ , ] . similarly, we did not observe a correlation between the hrv viral load and clinical features or laboratory data. our results suggest that the difference in disease severity may be more related to the host's susceptibility to hrv than to the viral load. viral load can vary according to the sampling process. therefore, the exact relationship between hrv viral load and disease severity merits further investigation. this study has several limitations. first, the study patients were limited to hospitalized children with lower respiratory tract infections at a single center. in addition, the cytokine levels detected in this study were low and the differences were overall minor.these factors may have affected our statistical results. in addition, an asymptomatic control group was not enrolled. including a control group is often a particular challenge in pediatric populations. despite these shortcomings, our results contribute to the understanding of hrv's role and immunopathogenesis in lower respiratory infection. hrv is an important pathogen of the lower respiratory tract in young children. in particular, hrv-a and hrv-c are the dominant species that cause respiratory difficulties including wheezing and chest retraction. hrv-b may cause minor lower respiratory infections in relatively older children. further studies are needed to clarify the pathogenesis of hrv, and to define the specific clinical roles of each species. supporting information s table. all relevant data, inclusive of particular epidemiologic and clinical data as well as cytokine profiles in hrv-infected children. (xlsx) human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia rhinovirus bronchiolitis and recurrent wheezing: -year follow-up evaluation of respiratory viral pathogens in acute asthma exacerbations during childhood detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis relationships among specific viral pathogens, virus-induced interleukin- , and respiratory symptoms in infancy rhinovirus illnesses during infancy predict subsequent childhood wheezing establishing a surveillance network for severe lower respiratory tract infections in korean infants and young children rhinovirus-associated hospitalizations in young children rhinoviruses are a major cause of wheezing and hospitalization in children less than years of age fatal respiratory infections associated with rhinovirus outbreak clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup role of rhinovirus c respiratory infections in sick and healthy children in spain human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease molecular epidemiology of human rhinovirus infections in kilifi, coastal kenya human rhinovirus species c infection in young children with acute wheeze is associated with increased acute respiratory hospital admissions patient characteristics and severity of human rhinovirus infections in children human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander prevalence of human rhinovirus in children admitted to hospital with acute lower respiratory tract infections in changsha human rhinovirus types and association with respiratory symptoms during the first year of life human rhinovirus species and season of infection determine illness severity novel human rhinoviruses and exacerbation of asthma in children biodiversity and clinicodemographic characteristics of human rhinoviruses from hospitalized children with acute lower respiratory tract infections in malaysia prospective evaluation of rhinovirus infection in healthy young children correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients rhinovirus load and disease severity in children with lower respiratory tract infections impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia the authors would like to thank ms. kim heui og for her technical assistance. key: cord- -b l epy authors: falsey, ann regina title: respiratory viral infections date: - - journal: genomic and precision medicine doi: . /b - - - - . - sha: doc_id: cord_uid: b l epy molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. polymerase chain reaction for the rapid and accurate diagnosis of viral infections has led to a better understanding of the epidemiology and impact of many common respiratory viruses and resulted in better patient care. over the past decade a number of new respiratory viruses including human metapneumovirus and new coronaviruses have been discovered using molecular techniques such as random primer amplification, pan-viral array and next generation sequencing. analysis of the host transcriptional response during respiratory viral infection using in-vitro, animal models and natural and experimental human challenge have furthered the understanding of the mechanisms and predictors of severe disease and may identify potential therapeutic targets to prevent and ameliorate illness. respiratory viruses are a major cause of morbidity and mortality throughout the world and affect persons of all ages [ ] [ ] [ ] [ ] . in addition to > million office visits for upper respiratory infections each winter, hospitals fill to capacity with admissions due to community acquired pneumonia, acute exacerbations of chronic obstructive pulmonary disease, asthma and bronchitis and many of these illnesses are due to viral infection. "pneumonia and influenza" consistently ranks as the fourth most common discharge diagnosis, and each year, , to , hospitalizations and to , deaths in the united states are attributable to influenza [ , [ ] [ ] [ ] [ ] . due to their epidemic nature influenza and rsv are widely recognized as pathogens in adults and children, respectively. however, the true burden of disease and the contributions of other viruses such parainfluenza viruses (piv), human metapneumoviruses (hmpv), coronaviruses (cov), and rhinoviruses (hrv) now being more fully recognized using modern molecular detection methods [ ] [ ] [ ] [ ] [ ] . in addition to sensitive and rapid diagnostic testing, new molecular techniques allow an understanding of viral evolution, mechanisms and predictors of severe disease, interrogation of vaccine responses, improved bacterial and viral diagnostics and associations of viral infections with non-respiratory medical events. in this chapter the many ways molecular and precision medicine have impacted the field of respiratory viral disease will be reviewed. in the past defining the epidemiology and impact of viral respiratory pathogens was significantly hampered by slow and/or insensitive diagnostic techniques such as cell culture and antigen detection [ , ] . polymerase chain reaction (pcr) has revolutionized the study of respiratory viruses and provides extremely sensitive, specific and rapid means for the detection of fastidious and non-cultivatable respiratory viruses [ ] . pcr based epidemiologic studies now provide a more complete understanding of the clinical spectrum and age ranges of populations affected [ ] [ ] [ ] [ ] [ ] [ ] . in one study, conventional methods yielded a viral diagnosis in % of pneumonia cases, while use of pcr increased the yield to % [ ] . technology has rapidly evolved from single-plex pcr and gel electrophoresis to multiplex real time assays where products are detected by luminescent signals proportional to the target amplified [ ] . there are currently a variety of commercially available assays that detect from to viral respiratory pathogens and maintain excellent sensitivity [ ] . many clinical microbiology laboratories are now moving to primarily molecular methods for viral detection and pcr formats are becoming increasingly simple so that nucleic acid extraction and pcr is fully automated with little operator input. molecular point of care assays will soon be feasible [ ] . in addition to providing more sensitive means of detecting known viruses, molecular methods are extremely useful for viral discovery [ ] [ ] [ ] . over the past several decades a number of new respiratory viruses or variants have been identified including hmpv, novel strains of coronaviruses (hku , nl , sars-cov, mers-cov), rhinovirus c, human boca virus, parechoviruses and new strains of avian influenza viruses. molecular methods have been critical for the rapid identification of new viruses associated with dramatic lethal outbreaks but also for pathogen discovery for routine respiratory illnesses. despite intensive investigation, in - % of lower respiratory illnesses no pathogen can be identified suggesting additional agents may yet be discovered [ ] [ ] [ ] . several different genomic approaches for pathogen discovery have been used successfully and include random primer amplification, pan-viral dna microarray and next generation sequencing ( fig. ) [ ] . if a viral class of an unknown pathogen or variant virus is suspected, consensus pcr using degenerate primers to detect sequences broadly conserved between members of a group can be used as was done to identify two new coronaviruses, hku and mers-cov [ , ] . another technique for viral discovery is random primer amplification with conventional shotgun sequencing of pcr products [ ] [ ] [ ] . such was the case when van den hoogen discovered a new respiratory virus in , in young children with bronchiolitis who tested negative for rsv [ ] . after detecting paramyxovirus like particles in cell culture, rna was subjected to random primer pcr and viral sequences were compared to all known pathogens. the new virus most closely aligned to avian pneumovirus but was determined to be a unique human pathogen and named human metapneumovirus (hmpv). similarly, in peiris identified a novel coronavirus as the cause of severe acute respiratory syndrome (sars-cov) using degenerate/random primers pcr amplification [ ] . using pan viral micro array investigators at the center for disease control and prevention independently identified the same sars-cov [ ] . in this technique, after random primer amplification, pcr products are hybridized to microarrays consisting of mer oligonucleotides derived from every fully sequenced viral genome. hybridized sequences are scraped from the microspot, amplified, cloned and finally sequenced [ ] . identification of completely novel infectious agents requires unbiased and sequence independent methods for universal amplification [ , , ] . conventional sanger sequencing may have poor sensitivity for genomes at low quantity. next generation sequencing (ngs) involves the analysis of millions of sequences and can detect small amounts of novel nucleic acid sequences in clinical samples. continuous sequences are assembled, host sequences are subtracted and the residual sequences are analyzed for similarity to known microbial sequences. ngs has led to the discovery a numerous novel human and animal pathogens [ ] . a recent study of nasopharyngeal aspirates of thai children with respiratory illness using ngs identified a number of mammalian viral sequences belonging to newly described families of viruses such anelloviridae as well as novel strains of hrv, enteroviruses and hbov [ ] . a critical step in viral discovery is the availability of bioinformatic tools to efficiently identify unique viral sequences in complex mixtures of host, bacterial and fungal sequences. new computational tools for analysis of the virome such as "virusseeker" are being developed [ ] . of note, detection does equate with causation and after discovery further studies are necessary to infer more than association. the genetic and antigenic evolution of error prone rna respiratory viruses, particularly influenza, has been of interest for several decades [ , ] . understanding the selective pressure exerted by pre-existing immunity on viral evolution may help design more effective influenza vaccines and surveillance of animal populations can be critical for early identification of emerging influenza viruses [ ] . advances in deep sequencing make it possible to measure low frequency within host viral diversity and factors such as antigenic diversity, antiviral resistance, and tissue specificity can now be studied to understand the complexities of viral evolution [ ] . influenza evolution at a population level has been studied years, yet, new antigenic variants are initially generated and selected at the level of the individual infected host. within a host, influenza viruses exist as a "swarm" of genetically distinct viruses [ ] . sanger sequencing defines consensus sequences and cannot resolve minority variants below % of the viral population. deep sequencing has been used in natural infection and human challenge studies to characterize between and within host genetic diversity [ , ] . the identification of low frequency mutations in the hemagglutinin (ha) antigenic sites or near the receptor-binding domain in vaccinated and unvaccinated influenza infected persons highlight viral evolution within a host due to selective immune pressure [ ] . similarly, ngs can reveal the rapid evolution of drug resistant variants during therapy [ ] . using samples collected over time, the mutational spectrum of h n influenza a virus in an immunocompromised child was delineated [ ] . individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages. the within host evolution of antiviral resistance reflected a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. within host sequencing diversity has also been examined in an infant with severe combined immune deficiency with persistent rsv infection [ ] . ngs was performed on samples obtained before and after bone marrow transplantation. the viral population appeared to diversify after engraftment with most variation occurring in the attachment protein (g). in addition, minority viral populations with palivizumab resistance mutations emerged after its administration. deep sequencing of hrv during human challenge studies has shown that hrv generates new variants rapidly during the course of infection with accumulation of changes in "hot spots" in the capsid, c, and c genes [ ] . a genome-wide association study (gwas) involves rapidly scanning sets of dna, or genomes, of many people to find genetic variations associated with a particular disease. typically, the genomes of cases are compared to non-affected controls and search for single nucleotide polymorphisms (snps) or polygenic changes that are associated with risk or protection from susceptibility or severity of the condition. gwas have been useful to find genetic variations and risk for asthma, cancer, diabetes, heart disease and autoimmune illnesses with relatively limited studies relating to infectious diseases [ , ] . recent studies examining host genetic factors conferring susceptibility to respiratory viruses such as pandemic h n influenza a, sars-cov and rsv now provide some insight into host genetic factors for respiratory viral infections [ ] [ ] [ ] . previously most influenza research focused on viral genetics of novel viruses, yet experience with h n and h n clearly indicate host factors also influence disease severity [ , ] . a number of candidate genes influencing respiratory virus susceptibility have been identified in animal and human studies and involve host virus interactions, innate immune signaling, interferon related pathways and cytokine responses (table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . over studies have evaluated genetic polymorphisms associated with severe rsv disease and none demonstrates dramatic results [ ] . most focused on one or a few candidate genes resulting in only modestly increased odds ratios of severe illness. a relatively large study of almost hospitalized children that examined snps in candidate genes demonstrated that susceptibility to rsv is complex with a several associations to a few innate immunity genes. these included a vitamin d receptor gene associated with down regulating interleukin (il- ), gamma interferon (ifn-γ), nitrous oxide synthase (nos a), the jun oncogene, an important transcriptional regulator for innate immune pathways, and ifn-α (ifna ) an antiviral cytokine [ ] . the host transcriptional response can be analyzed to investigate disease pathogenesis using a variety of methods including in-vitro studies of bronchial epithelial cells (bec), animal models and infection both natural and experimental challenge [ ] [ ] [ ] [ ] . in addition, two compartments, the respiratory epithelium and blood can be sampled in human studies and interrogated using different viruses or viral strains to develop gene signatures for prognosis, as indicators of severity and to identify potential therapeutic targets. most respiratory viral mechanistic studies have been performed using influenza viruses, rsv, hrv and coronaviruses [ ] [ ] [ ] [ ] . using bec, the common and (h n , h n , h n and h n ) and analyzed cellular responses using microarray [ ] . common proinflammatory cytokines and antigen presentation were identified although each viral response was unique and notably, h n responses were most similar to h n . the response of different clinical isolates of rsv in a cells, and monocyte derived human macrophages demonstrated that the pattern of innate immune activation was both host cell and viral strain specific [ ] . using rna seq, differences in il- and ccl were noted among the responses to different clinical isolates suggesting different rsv strains may vary in inherent virulence. human studies have shown significant differences in the blood transcriptional profiles which change over time and differ depending on the infecting respiratory virus. mejias and colleagues were able to differentiate rsv, hrv and influenza in young children based on the blood gene profile (fig. ) . hrv infection exhibited the mildest innate and adaptive responses compared to rsv and influenza and neutrophil gene expression was greatest in rsv infection with marked suppression of b and t cell and lymphoid responses [ ] . notably, gene expression changes persisted up to month after infection. similarly, studies of h n infected patients showed transcriptional profile changes persisting up to month with a transition from innate to adaptive immunity [ ] . because of the association of hrv and exacerbations of asthma, the host response to hrv has been of particular interest [ ] [ ] [ ] [ ] . studies using becs from asthmatic and healthy donors demonstrate different transcriptional profiles when infected with hrv [ ] . hrv, similar to other picornaviruses induces gene expression down regulation by the a and c proteins. in both asthmatic and healthy control derived cells the majority of genes were down regulated after exposure to hrv. however, some significant expression differences in inflammatory, tumor suppressor, airway remodeling and metallopeptidase pathways have been noted in asthmatic derived cells. asymptomatic hrv infection is quite common and its role in asthma pathogenesis has been questioned. interestingly, heinonen et al. did not find a difference in the blood transcriptome of asymptomatic hrv infected children compared to non-infected controls [ ] . whereas, wesolowska-anderson and colleagues demonstrated over differentially expressed genes in the nasal epithelium of asymptomatic infected hrv patients [ ] . thus, the blood transcriptome may not be as informative as the nasal epithelial transcriptional response for asymptomatic hrv infection. given the significant host response to asymptomatic infection, hrv may play a role in asthma exacerbations in the absence of clinically evident disease. lastly, it may be possible to identify patients with asthma who are prone to frequent hrv related exacerbations by examining the gene expression response of their pbmcs stimulated with hrv [ ] . gene expression studies focusing on illness severity may enhance our understanding of disease pathogenesis, can identify potential therapies to modulate harmful host responses and can be used to develop biomarkers for predicting life threatening disease [ , [ ] [ ] [ ] . a number of studies have been undertaken to understand the pathogenesis of severe rsv in young children and have identified a variety of gene expression patterns in blood including under expression of t cell cytotoxicity/nk cells and plasma cell genes, as well as upregulation of jak/stat, prolactin, il- signaling, cell to cell signaling, and immune activation pathways [ , ] . using nasal epithelial gene expression analysis, van den kieboom identified differentially expressed genes in children with mild, moderate and severe rsv infection [ ] . ubiquitin d, tetraspanin , mucin , β microseminoprotein, chemokine ligand were up regulated and differentiated mild from severe illness. lastly, nasal gene expression is complicated by interactions of the nasal microbiota and host cell gene responses [ ] . in nasal samples from children with rsv infection, h. influenzae and s. pneumoniae dominated microbiota, toll like receptors and neutrophil/macrophage signaling were over expressed and the presence of h. influenzae and s. pneumoniae along with age and sex were predictive of risk of hospitalization due to rsv. transcriptional profiling related to severity has been analyzed in seasonal influenza as well as emerging avian pathogens with a recognition that disease is not only due to an infection with a novel virus in a non-immune host but may also be due to an exaggerated host immune response [ , ] . in a study of primarily seasonal influenza (h n , h n ), influenza infection was associated with a significantly stronger antiviral, cytokine, attenuation of t/nk cell response compared to patients with respiratory illnesses of unknown etiology regardless of severity [ ] . notably, ifn and ubiquitination was significantly down regulated in those with severe vs. mild to moderate disease. in a study of the lethality of h n influenza and h n vietnam influenza virus in macaques, upregulation of key components of the innate immune response and cell death pathways were noted were noted with h n infection but were down regulated with h n [ ] . early up regulation of the inflammasome likely resulted in some of the severe tissue damage noted with the h n influenza infections. in vitro, animal and human challenge studies have been used to identify new strategies control or prevent symptomatic or severe infection [ , ] . in hrv challenge studies, virperin expression correlated with rhinorrhea and chilliness. knockdown of expression resulted in increased viral replication in becs suggesting virperin has antiviral actions and might have potential therapeutic use. influenza challenge studies clearly show a definable transcriptomic profile in the blood prior to the onset of symptoms offering the possibility of earlier and more effective oseltamivir treatment [ , ] . lastly, host gene expression studies may allow investigation into links between respiratory viral infections and specific non-respiratory events. there is ample epidemiologic evidence that influenza epidemics are linked with increased rates of strokes and myocardial infarction (mi) [ , ] . increased rates of falls and functional decline in nursing homes have also been associated with increased influenza activity [ , ] . however, direct links of events to viral infection are scarce in part due the event of interest may follow the infection by several weeks when the virus is no longer detectable by traditional testing. several gene profiling studies have identified viral infection signatures that may persist up to -month post infection [ , ] . thus, it might be possible to study patients with falls or cardiac events for evidence of recent viral infection using a host response viral signature. in addition, evaluating the host response can provide information on mechanisms of disease. a viral gene signature was used to evaluate patients undergoing cardiac catheterization [ ] . notably, % vs. %, p = . of those with a viral gene signature present vs. those without viral signatures, suffered an mi. furthermore, h n infected patients showed an increased gene platelet expression signature providing insight into how infection may induce a prothrombotic state. given the availability of rapid and accurate multiplex pcr for viral detection, host-based diagnostics might seem unnecessary. however, current pcr assays use conserved known viral sequences but can miss novel or significantly mutated viruses. this issue was seen in with pandemic h n when influenza pcr assays had to be adapted to optimally detect the new influenza strain [ ] . the emergence of novel respiratory viruses are a persistent threat and methods to detect a "viral signature" in the setting of clusters of severe pneumonia cases could be very useful. zaas and colleagues developed an acute respiratory viral gene signature using microarray analysis of the blood from volunteers experimentally infected with influenza a, hrv or rsv [ ] . the signature was subsequently % sensitive and % specific in classifying as viral influenza and hrv infected patients presenting to an emergency room. additionally, a distinct blood transcriptome signature was noted in patients with severe h n pneumonia [ ] . upregulated genes included those related to cell cycle, dna damage, apoptosis, protein degradation, and t helper cells. down regulated genes were primarily in immune response pathways suggesting immunosuppression as a mechanism of severe influenza pneumonia. investigators developed a gene classifier which predicted h n influenza a regardless of concomitant bacterial infection and such a predicator could guide antiviral therapy in the face of negative pathogen detection methods. in most cases of respiratory infection, the precise microbial etiology is unknown and antibiotics are frequently administered empirically [ , ] . although sensitive molecular diagnostics (pcr) now allow rapid diagnosis of a wide variety of respiratory viruses, their impact on patient management and antibiotic prescription has been modest primarily due to concern about bacterial co-infection [ ] [ ] [ ] . approximately % of adults hospitalized with a documented viral respiratory infection have evidence of concomitant bacterial infection and thus clinician concerns are reasonable [ ] . importantly, sensitive and specific diagnostic tests for bacterial lung infection are currently lacking [ , ] . although the site of infection is the respiratory tract, blood is a convenient sample comprised of components of the innate immune system (neutrophils, natural killer cells), as well as the adaptive immune system (b and t lymphocytes) [ ] . recent studies indicate that viral and bacterial infections trigger pathogen specific host transcriptional patterns in blood, yielding unique "bio-signatures" that may discriminate viral from bacterial causes of infection [ ] [ ] [ ] [ ] . in the largest study to date, tsalik et al. used gene expression in blood to discriminate bacterial from viral infection or non-infectious illness in subjects with respiratory illness [ ] . these investigators defined predictor genes in a model with an accuracy of % to discriminate clinically adjudicated bacterial, viral, and non-infectious illness. most studies to date have used micro array but recently rnaseq has been used to differentiate viral and bacterial respiratory illness and in one study genes were noted to be differentially expressed [ ] . three pathways (lymphocyte, α-linoleic acid metabolism, igf regulation pathways) which included genes as predictors for bacterial infection from non-bacterial infection (naïve auc = . ; nested cv-auc = . ). to date, a number of gene expression studies of adults and children have developed predictors with similar accuracy (auc ranging from % to %), yet there has been little overlap in classifying genes identified [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . diverse populations, types of infection, plus alternate analytic tools used, likely explain the different genes identified. more work needs to be done to refine predictive gene sets including patients with mixed viral-bacterial respiratory tract infection. most studies to date have focused on blood; however, analysis of the nasal respiratory epithelium which is the site of infection might offer advantages. although data are limited, several recent papers demonstrate that nasopharyngeal host response can also be used as a diagnostic for respiratory viruses [ , , ] . immune response to influenza vaccine is variable and influenced by a variety of factors including prior vaccinations and infections, age, the presence of underlying conditions and the type of vaccine administered. yet, even among a relatively homogeneous cohort of young healthy adults, antibody responses to vaccine can be variable [ ] . transcriptional profiling of whole blood provide insights into the mechanisms of variability, the effects of age, and vaccine types. the ability to predict vaccine response at baseline based on a transcriptomic signature would have significant clinical implications. to understand the biologic effects of live attenuated influenza vaccine (laiv) compared to trivalent inactivated vaccine (tiv) blood transcription profiles from young children were assessed by microarray at day post vaccination [ ] . many more genes were differentially expressed in children receiving laiv compared to tiv ( vs. , respectively) and many modulated type ifn. the efficacy of laiv has been problematic in recent years and assessing stimulation of type ifn genes could represent a potential biomarker for response to laiv [ ] . bucasas and colleagues evaluated gene expression at multiple time points after vaccination of healthy young men with tiv [ ] . they noted marked up regulation of gene expression of ifn signaling, il- regulation, antigen processing and presentation genes within h of vaccination and were able to define a gene expression signature that correlated with the magnitude of antibody response. in another study, a gene profile predictive of antibody response days after influenza vaccination of young and older adults was developed [ ] . notably, the predictive genes were the same in young and old as well as a subgroup of subjects with diabetes suggesting similar pathways were involved despite differences in age and underlying medical conditions. additionally, transcriptional profiling has been used to signatures in blood associated with b cell memory responses to vaccination. in a study of older and middle aged adults vaccinated with tiv including an h n antigen, metabolic, cell migration/adhesion, map kinase and nf-ᵏb cell genes correlated with peak memory b cell elispots [ ] . finally, in a study of over subjects vaccinated over several seasons, a predictive signature of nine genes and three gene modules were significantly associated with the magnitude of the serum antibody response (fig. ) [ ] . interestingly and in contrast to a previous study, the signature was distinct to the younger cohort. for example, inflammatory genes were associated with better response in the young but a worse response in the elderly. in summary, gene expression studies could be used to evaluate new vaccines and develop predictors of vaccine response in different subgroups of patients based on age and disease state allowing for individualized vaccine regimens. molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. the ability to accurately diagnosis viral infections has not only impacted patient care but also changed our perceptions of the burden of disease and populations effected. transcriptional profiling of blood and nasal epithelium may provide therapeutic targets to prevent and ameliorate illness as well as offer predictors of severe disease. ( ) .) summary health statistics for u.s. children: national health interview survey global respiratory syncytial virus-associated mortality in young children (rsv gold): a retrospective case series mortality associated with influenza and respiratory syncytial virus in the united states epidemiology of viral respiratory infections epidemiology of influenza trends in hospitalizations for pneumonia among persons aged years or older in the united states hospitalizations associated with influenza and respiratory syncytial virus in the united states estimating influenza-associated deaths in the united states impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection human metapneumovirus infections in adults: another piece of the puzzle human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease update on human rhinovirus and coronavirus infections detection of respiratory viruses by molecular methods molecular detection of respiratory viruses respiratory syncytial virus infection in elderly and high-risk adults etiology of communityacquired pneumonia: increased microbiological yield with new diagnostic methods polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia incidence and characteristics of viral community-acquired pneumonia in adults molecular epidemiology of rhinovirus detections in young children human metapneumovirus in the preterm neonate: current perspectives viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation profile of the alere i influenza a & b assay: a pioneering molecular point-of-care test viral surveillance and discovery viral pathogen discovery viral discovery and sequence recovery using dna microarrays population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children community-acquired pneumonia requiring hospitalization respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia isolation of a novel coronavirus from a man with pneumonia in saudi arabia a newly discovered human pneumovirus isolated from young children with respiratory tract disease coronavirus as a possible cause of severe acute respiratory syndrome cloning of a human parvovirus by molecular screening of respiratory tract samples a novel coronavirus associated with severe acute respiratory syndrome exploring the potential of next-generation sequencing in detection of respiratory viruses virusseeker, a computational pipeline for virus discovery and virome composition analysis mapping the antigenic and genetic evolution of influenza virus integrating influenza antigenic dynamics with molecular evolution molecular evolution and emergence of h n avian influenza virus in central china within-host evolution of human influenza virus deep sequencing reveals potential antigenic variants at low frequencies in influenza a virus-infected humans deep sequencing of influenza a virus from a human challenge study reveals a selective bottleneck and only limited intrahost genetic diversification presence of oseltamivir-resistant pandemic a/h n minor variants before drug therapy with subsequent selection and transmission intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment, and natural selection within-host whole-genome deep sequencing and diversity analysis of human respiratory syncytial virus infection reveals dynamics of genomic diversity in the absence and presence of immune pressure rhinovirus genome evolution during experimental human infection genome-wide association studies and susceptibility to infectious diseases host-pathogen interactions revealed by human genome-wide surveys immunogenetic factors associated with severe respiratory illness caused by zoonotic h n and h n influenza viruses the role of polymorphisms in host immune genes in determining the severity of respiratory illness caused by pandemic h n influenza human genetic factors and respiratory syncytial virus disease severity chemokine receptor big up tri, open allele in patients with severe pandemic (h n ) contrasting effects of ccr and ccr deficiency in the pulmonary inflammatory response to influenza a virus enrichment of variations in kir dl /s and kir dl /l among h n / icu patients: an exploratory study ifitm restricts the morbidity and mortality associated with influenza the lower serum immunoglobulin g level in severe cases than in mild cases of pandemic h n influenza is associated with cytokine dysregulation genetic variants associated with severe pneumonia in a/h n influenza infection associations of functional nlrp polymorphisms with susceptibility to food-induced anaphylaxis and aspirininduced asthma gene polymorphisms in the nalp inflammasome are associated with interleukin- production and severe inflammation: relation to common inflammatory diseases? the magnitude and specificity of influenza a virus-specific cytotoxic t-lymphocyte responses in humans is related to hla-a and -b phenotype mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannosebinding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection significance of the myxovirus resistance a (mxa) gene - c>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection association of sars susceptibility with single nucleic acid polymorphisms of oas and mxa genes: a case-control study polymorphisms of interferon-inducible genes oas- and mxa associated with sars in the vietnamese population genetic variation in oas is a risk factor for initial infection with west nile virus in man a missense mutation of the toll-like receptor gene in a patient with influenza-associated encephalopathy genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes genomewide association analysis of respiratory syncytial virus infection in mice genes associated with rsv lower respiratory tract infection and asthma: the application of genetic epidemiological methods to understand causality unusual haplotypic structure of il , a susceptibility locus for a common respiratory virus variants of the chemokine receptor ccr are associated with severe bronchiolitis caused by respiratory syncytial virus association of respiratory syncytial virus bronchiolitis with the interleukin gene region in uk families genetic variation at the il gene locus is associated with severity of respiratory syncytial virus bronchiolitis a functional variation in cd increases the severity of pandemic h n influenza a virus infection using gene expression profiles from peripheral blood to identify asymptomatic responses to acute respiratory viral infections transcriptomics in human challenge models lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection gene expression patterns induced at different stages of rhinovirus infection in human alveolar epithelial cells nasopharyngeal gene expression, a novel approach to study the course of respiratory syncytial virus infection mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury transcriptomic characterization of the novel avian-origin influenza a (h n ) virus: specific host response and responses intermediate between avian (h n and h n ) and human (h n ) viruses and implications for treatment options pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses distinct patterns of innate immune activation by clinical isolates of respiratory syncytial virus clinical correlations of transcriptional profile in patients infected with avian influenza h n virus rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma how rhinovirus infections cause exacerbations of asthma a genome-byenvironment interaction classifier for precision medicine: personal transcriptome response to rhinovirus identifies children prone to asthma exacerbations dual rna-seq reveals viral infections in asthmatic children without respiratory illness which are associated with changes in the airway transcriptome rhinovirus detection in symptomatic and asymptomatic children: value of host transcriptome analysis association of dynamic changes in the cd t-cell transcriptome with disease severity during primary respiratory syncytial virus infection in young infants host transcription profile in nasal epithelium and whole blood of hospitalized children under years of age with respiratory syncytial virus infection gene expression signatures as a therapeutic target for severe h n influenza-what do we know so far? nasopharyngeal microbiota, host transcriptome, and disease severity in children with respiratory syncytial virus infection patient-based transcriptome-wide analysis identify interferon and ubiquination pathways as potential predictors of influenza a disease severity gene expression profiles during in vivo human rhinovirus infection: insights into the host response a genomic signature of influenza infection shows potential for presymptomatic detection, guiding early therapy, and monitoring clinical responses influenza infection and risk of acute myocardial infarction in england and wales: a caliber self-controlled case series study acute myocardial infarction after laboratory-confirmed influenza infection effect of influenza on functional decline a study of the impact of influenza on the functional status of frail older people gene expression profiles link respiratory viral infection, platelet response to aspirin, and acute myocardial infarction the current epidemiology and clinical decisions surrounding acute respiratory infections gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans a distinct influenza infection signature in the blood transcriptome of patients with severe communityacquired pneumonia appropriate antibiotic use for acute respiratory tract infection in adults treating viral respiratory tract infections with antibiotics in hospitals: no longer a case of mistaken identity bacterial complications of respiratory tract viral illness: a comprehensive evaluation routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial diagnostic and prognostic accuracy of clinical and laboratory parameters in community-acquired pneumonia developing molecular amplification methods for rapid diagnosis of respiratory tract infections caused by bacterial pathogens assessing the human immune system through blood transcriptomics superiority of transcriptional profiling over procalcitonin for distinguishing bacterial from viral lower respiratory tract infections in hospitalized adults gene expression patterns in blood leukocytes discriminate patients with acute infections gene expression profiles in febrile children with defined viral and bacterial infection robust classification of bacterial and viral infections via integrated host gene expression diagnostics host gene expression classifiers diagnose acute respiratory illness etiology transcriptomic biomarkers to discriminate bacterial from nonbacterial infection in adults hospitalized with respiratory illness diagnostic test accuracy of a -transcript host rna signature for discriminating bacterial vs viral infection in febrile children association of rna biosignatures with bacterial infections in febrile infants aged days or younger a host-based rt-pcr gene expression signature to identify acute respiratory viral infection detection of host response to viral respiratory infection by measurement of messenger rna for mxa, trim , and viperin in nasal swabs antiviral response in the nasopharynx identifies patients with respiratory virus infection integrative genomic analysis of the human immune response to influenza vaccination a whole genome transcriptional analysis of the early immune response induced by live attenuated and inactivated influenza vaccines in young children early patterns of gene expression correlate with the humoral immune response to influenza vaccination in humans systems biology of vaccination for seasonal influenza in humans transcriptional signatures of influenza a/h n -specific igg memory-like b cell response in older individuals multicohort analysis reveals baseline transcriptional predictors of influenza vaccination responses key: cord- -lbx plqx authors: wang, wei; cavailler, philippe; ren, peijun; zhang, jing; dong, wei; yan, huajie; mardy, sek; cailhol, johann; buchy, philippe; sheng, jun; fontanet, arnaud; deubel, vincent title: molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: lbx plqx background: numerous viruses are responsible for respiratory infections; however, both their distribution and genetic diversity, in a limited area and a population subgroup, have been studied only rarely during a sustained period of time. methods: a -year surveillance program of children presenting with acute respiratory infections (aris) was carried out to characterize the viral etiology and to assess whether using gene amplification and sequencing could be a reliable approach to monitor virus introduction and spread in a population subgroup. results: using multiplex rt-pcr, different respiratory viruses were detected within the nasopharyngeal positive samples collected among children aged < -year old who presented with ari during october to september . a single virus was detected in patients ( . %), and two to four viruses in patients ( . %). the most frequent causative viruses were respiratory syncytial virus (rsv) ( . %), human bocavirus ( . %), and human rhinovirus (hrv) ( %). rsv was more prevalent in winter and among young infants. cases of seasonal influenza a and b viruses were reported mainly in january and august. an increase in adenovirus infection was observed during the spring of the second year of the study. sequence analyses showed multiple introductions of different virus subtypes and identified a high prevalence of the newly defined hrv-c species. a higher viral incidence was observed during the winter of , which was unusually cold. conclusions: this study supports the usefulness of multiplex rt-pcr for virus detection and co-infection, and for implementation of a molecular monitoring system for endemic and epidemic viral respiratory infections. since the epidemic of severe acute respiratory syndrome (sars) in , and the recent attention on possible influenza pandemics, sustained surveillance project was required to detect endemic, epidemic and newly emerging respiratory pathogens. the diagnosis of respiratory viruses mainly relies on molecular techniques. multiplex rt-pcr (mrt-pcr) techniques allow identification of a majority of respiratory viruses [ ] [ ] [ ] as well as co-infections. , in the present -year study, we used a five-tube mrt-pcr assay we implemented in the pasteur institute network in the asian region (http://www.pasteur-international.org/ip/easysite/ pasteur-international/activites-scientifiques/projets/tous-lesprojets/sisea), which covered common respiratory viruses, to identify viruses in nasopharyngeal specimens in children with table criteria of patient enrollment. children younger than -year old, first onset within h, subjects already under antiviral treatment for any prophylactic or curative purpose and fever (t ≥ • c) plus cough and/or sore throat and/or dyspnoea or tachypnea, cyanosis, cough, pleuritic chest pain, hypoxemia and signature of the patient consent agreement ari. sequencing primers specific to other fragments of viral genes were employed to amplify the positive samples for sequencing and phylogenetic analysis. the sequences showed the genetic variation of viruses circulating in the region, and identified the virus evolution and introduction of new variants. this study allowed cartography of viral etiology of a large panel of viruses that were co-circulating in ari in children in a district of shanghai, with the aim of implementing a monitoring system for endemic or epidemic viral respiratory infections. between october , and september , , all subjects aged < years, who presented with an ari syndrome and attended the outpatient ward of the pediatric department of shanghai nanxiang hospital, china, were enrolled prospectively. the protocol was approved by the ethical committee of shanghai nanxiang hospital and by the biomedical committee of institut pasteur in paris. written informed consent of a parent or a legal guardian was required. ari was defined as the presence of fever (at least • c) plus cough and/or sore throat (table ) . study enrollment was organized twice weekly (every monday and thursday); all patients who consulted on monday and thursday and presented with the above case definition were included in the study. upon enrollment, systematic recordings were made of the patients' demographic characteristics and medical history using a standardized questionnaire. the questions included detailed signs and symptoms, laboratory and radiology examinations; the presence of a chronic underlying disease; and family smoking history. after a complete physical examination, the children were classified into three different disease groups on the basis of signs and/or symptoms indicating the inflammation site: upper respiratory tract infection, bronchitis and pneumonia. patients and their parents were interviewed by the same doctor to obtain demographic data and information about their clinical presentation. for each ari case, nasopharyngeal swabs (npss) were obtained by the same nurse. specimens were collected with sterile cottontipped swabs that were introduced into the nostril and the pharyngeal areas, and then placed in ml viral transport medium. the npss were then transported at • c to the virology department of the institut pasteur of shanghai, where they were divided into aliquots, and stored at − • c. rna extraction-total rna from nps aliquots was extracted using a qiaamp viral rna minikit (qiagen, hilden, germany) in accordance with the manufacturer's protocol. purified rna was frozen at − • c in aliquots. multiplex rt-pcr-a mrt-pcr previously published was improved and employed in this study for virus detection. it was initially described by bellau-pujol et al. ( ) for virus identification including iav, ibv, icv, rsv, hmpv, piv - , hcov-oc and e, and hrv in three tubes with hemi-nested pcr then later improved by vabret et al. for viruses plus with hcov-hku and hcov-nl in four tubes. besides, the primers used for hrv detection could also detect hev, and the two viruses were then differentiated based on the sizes of the amplified products. the dna band with the size of - bp corresponded to rhinovirus while the band with the size of - bp corresponded to enterovirus. hence, the mrt-pcr could detect viruses. the mrt-pcr multiplex and the hemi-nested multiplex detected and . tcid of rsv a, respectively, and . and . tcid of influenza virus a/h n , respectively. we have improved the method to detect viruses in a fivetube mrt-pcr assay by introducing specific primers to adv and hbov. , moreover, we eliminated previous hemi-nested pcr step to avoid cross-contamination but introduced sequencing of amplification products. the assay was compared with commercialized resplex ii assay (qiagen) in previous study and its sensitivity reached . tcid of rsv b, . tcid of influenza virus a/h n , and . tcid of ibv, respectively. tube targeted iav, ibv, rsv, hmpv; tube , piv - ; tube , hrv and icv; and tube , hcov- e, hcov-oc , hcov-nl and hcov-hku . , , tube targeted adv and hbov using previously published primers: hbov ( f, -gasctctgtaagtactattac- ; r, -ctctgtgttgactgaatacag- ); adv (adhex f, -caacacctaygastacatgaa- ; adhex r, -acatccttbckgaagttcca- ). , rna was amplified using a one-step rt-pcr kit (qiagen) as previously described. in brief, . l of extracted rna was mixed with a × buffer and . mm dntp, . m of each primer, and l l of enzyme mix, and depctreated ultrapure water was added to a final volume of l. amplification programs included reverse transcription at • c for min, inactivation at • c for min, followed by cycles at • c for s, • c (tubes , and ) or • c (tubes and ) for s, • c for s and final extension at • c for min. the amplified dna products were detected by . g/l ethidium bromide/ % agarose gel electrophoresis. cloning and sequencing-sequencing primers were designed based on the conserved region of each virus or on previous publications [ ] [ ] [ ] [ ] [ ] [ ] (table ) . extracted rna was amplified by specific monoplex rt-pcr following the same protocol as above, but with a hybridization temperature of • c. the dna products were purified from agarose gels using qiaquick gel extraction kit (qiagen), and were ligated into pmd -t vector (takara biotechnology, dalian, china). recombinant plasmids were sequenced by biosune sequence company and invitrogen biotechnology company in shanghai, china. phylogenetic analysis-multiple sequences were aligned using clustal x (v . ). the multiple-sequence alignment was subjected to phylogenetic analyses using programs in the phylip package (v . ). bootstrap analysis was performed using seqboot, in which replicate number was . dnadist and neighbor were used to obtain a distance matrix; in dnadist, the transition/transversion ratio was . consensus trees were computed by consense, and then rerooted with retree. the final tree was visualized and edited with mega version . the patient data were analyzed using statistical package for the social sciences (spss) for window version . (spss inc. chicago, a viral etiology could be determined in of the ( . %) patients. multiple viral infections were detected in ( . %) patients ( with two pathogens, with three pathogens, and one with four pathogens) ( table ). among virus-positive cases, ( . %) were diagnosed by clinicians as bronchitis, ( . %) as pneumonia, and ( . %) were diagnosed with upper respiratory tract infection. overall, viral pathogens were detected: rsv was the most frequent pathogen (n = , . %). the second to the fifth most frequent pathogens were hbov (n = , . %); iv (iav and ibv) (n = , . %); piv , or (n = , . %), and hrv (n = , . %). hmpv (n = , . %), adv (n = , . %), hcov-oc , e, nl or hku (n = , . %). hev (n = , . %) were also detected occasionally. among the patients diagnosed with a multiple viral infection, the most frequent pathogens were hbov, rsv, hrv and adv (data not shown). the monthly distribution of viruses is presented in figs. and . viruses were detected significantly more often during fall or winter than during other seasons ( % and %, respectively, p < . ). rsv and hbov identifications were most frequent in the fall and winter. iv showed a biannual distribution, one peak in winter and another in the summer. patients included in this study were aged from month to years. the median age was years. rsv was more frequent in younger children (p = − ) with out of virus-positive cases from months to years age group. no iav or ibv infection was detected in children younger than -year old (table ). only patients from to months age group were enrolled in the study. to identify the prevalent subtype of different viruses and the similarity of the virus strains, virus-positive samples were sequenced for target genes, when the amount of genetic material amplified was sufficient (table ) , and phylogenetic trees were constructed (data not shown; dendrograms are available on request to table . in sequenced adv strains, were species b ( . %) and three were species c ( . %). most of them were similar to serotype adv- (data not shown). in hrv strains, were species a ( . %), five were species b ( . %), and were species c ( . %). hrv showed high variation in nucleotide identity ( - % in hrv-a, - % in hrv-b, and - % in hrv-c) as previously described (huang et al.) . among iav strains, were seasonal h n ( - % nucleotide identity in the ha gene) and were seasonal h n ( - % identity) strains. in out of ibv strains, ha and na gene fragments were sequenced, and comparison showed that the ibv strains were more conserved in the na gene ( - % nucleotide identity) than ha gene ( - % identity). only out of hbovs were sequenced but showed high nucleotide identity in the st gene as previously described. the sequences of hcovs were highly conserved ( - % nucleotide identity in hcov-nl , % in hcov- e). in hmpv strains, were genotype b ( %), were genotype b ( %), and seven were genotype a ( . %) with high nucleotide identity ( - % in a and - % in b and b ). only out of rsvs were sequenced and nucleotide sequence analysis of a glycoprotein gene fragment showed all of the strains were classified into rsv a subtype (data not shown). the results indicated a low variability of these viruses that circulated in the region during the -year period. among virus-positive cases, ( . %) patients presented with high fever (> • c) and cough, ( . %) patients with dys- response may be linked to a bacterial infection. no specific virus was associated to polynucleosis. from october to september , outpatients aged from month to years were included in a surveillance program of viral etiology in ari. less than % of the children enrolled in this outpatient study were aged of less than months, suggesting that very young children may show more severe symptoms and hospitalized. fifteen different viruses were detected in samples ( . %). thus, respiratory viruses were the major pathogens responsible for ari in children in shanghai and multi-infections of different viruses ( . %) were frequently observed. although serotype identification is critical for epidemiological surveillance, the serotyping is time consuming and costly, and limited due to cross-reactivity of the tests. we sequenced the fragments of genes coding for virus antigenic proteins and analyzed by phylogenetic analysis the sequence diversity to monitor the molecular evolution of circulating virus. during the outbreak of adv from march to june in , the majority of strains ( out of ) was adv-b species and of serotype adv- . in adv-b species, only serotype adv- was reported to cause severe infection, which may partly explained that in our study adv-infected patients showed only mild symptoms. the iav strains detected during the period from january to april were mainly h n but the strains detected from july to september were mainly h n . this suggests that subtype h n replaced the h n subtype and predominated during the next year in the region. as vaccination for seasonal influenza (iav h n , h n and ibv) was not included in the children's routine vaccination program in shanghai, and was usually based on the strains circulating worldwide in the precedent summer, the local population was not protected against the new subtype that emerged in the summer of . hrv is classified into three species: hrv-a, hrv-b and hrv-c by phylogenetic analysis based on sequences of vp gene and/or utr. [ ] [ ] [ ] a predominance of the newly identified species hrv-c ( . %) and the recombinant strains were observed based on which two new subspecies of hrv-c were proposed as hrv-ca and hrv-cc. this suggests the emergence of new variant strains of hrv in future that might cause a new epidemic. eleven hev were detected in the study but were not analyzed further. hmpv is another recently identified respiratory virus and has been found worldwide. it is grouped into four distinct genetic lineages based on the f gene: a , a , b , and b . in this study, two strains of hmpv-a , strains of hmpv-b and two strains of hmpv-b were detected in the first season, whereas one strain of hmpv-a , three strains of hmpv-b and strains of hmpv-b were identified in the second season. no hmpv-a lineage was found. hence, a change of predominant lineage in the seasons was observed, but no association between the severity of infection and genetic drift of hmpv was found, as shown in other previous studies. , besides, studies showed more sequence diversity in g and sh genes but not in f gene, which could explain the constant incidence of hmpv infection in the population. hcov-hku (in group ii with hcov-oc ) and hcov-nl (in group i with hcov- e) are two novel coronaviruses. , during the present -year study, hcov-nl and hcov- e were the major hcov circulating in shanghai, whereas only one strain of hcov-oc and one of hcov-hku were detected, indicating a sporadic introduction of group ii hcov to the region. in context that the recent emerged hcov, like hku- and sars whose sequence is more homologous to group ii virus, could cause severe respiratory infection, the surveillance for emergence of new species of hcov is necessary. the co-infection of rsv and hbov was frequently detected among the samples, whereas these two viruses co-dominated in cold season. hbov was the second most prevalent virus ( . %), and the co-infection rate of hbov with other respiratory viruses was . %, compared to % in non-hbov-infected patients. this was lower than the co-infection rate reported previously, which ranged up to %. [ ] [ ] [ ] [ ] a previous study showed that hbov increased the severity of bronchiolitis in children less than -year-old co-infected with rsv, and that it is not an occasional virus. however, no correlation of hbov infection with clinical severity was observed in this and its related study. one study carried out in wuhan, china analyzed peripheral blood samples by indirect immunofluorescence to detect rsv, iav, ibv, adv, piv - , chlamydia pneumonia and mycoplasma pneumonia in children ari inpatients and used viremia as sign of severe infection. it showed that % of cases were co-infected by multiple agents and iav, ibv and piv were associated with co-infection. in addition, studies showed up to % of co-infection in hospitalized children and rsv co-infection was associated with clinical severity. , however, no such correlation was found in this study. this may be due to differences in the criteria of patient enrollment and in the lower severity of clinical signs observed. bacteria-virus co-infection was commonly found in inpatients. in our study, only polynucleosis (> , on absolute count) was considered as a sign of bacterial infection and was observed in patients infected with a respiratory virus. hence, future studies should focus on severe respiratory infection to identify viral determinants of disease severity and should introduce bacteriological test. up to specimens were negative in mrt-pcr, despite all of them matched well with the inclusion criteria for ari. negative results could have resulted from the low load of viral material in samples, or to infection with bacteria instead of virus. new sensitive tools such as mass-tag or high-throughput sequencing have been developed recently to identify new viruses and bacterial pathogens. implementation of these new molecular techniques for samples that are negative in mrt-pcr might be considered in the future. this is believed to be the first study in china to characterize common respiratory viruses in pediatric ari during a -year consecutive period in a limited community with an important immigrant population. using mrt-pcr followed by sequencing and phylogenetic analysis, we could identify a wide variety of agents and differentiate highly pathogenic viruses from less virulent seasonal respiratory viruses. the sequence analysis result could be useful to improve the primer design for rt-pcr and to identify new subtype virus, for example hrv-c. it monitored sustaining virus circulation in the community, which could serve as a baseline of the annual distribution of viruses for surveillance of unusual prevalence of one specific virus. bronchiolitis-associated hospitalizations among us children trends in infectious disease mortality in the united states during the th century human rhinoviruses: the cold wars resume human bocavirus and acute wheezing in children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand serodiagnosis of human bocavirus infection correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection development of three multiplex rt-pcr assays for the detection of respiratory rna viruses direct screening of clinical specimens for multiple respiratory pathogens using the genaco respiratory panels and comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-pcr assays human (non-severe acute respiratory syndrome) coronavirus infections in hospitalised children in france cloning of a human parvovirus by molecular screening of respiratory tract samples molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium human metapneumovirus infection in hospitalized children with acute respiratory disease in korea detection and characterization of new influenza b virus variants in cocirculation and evolution of two lineages of influenza b viruses in europe and israel in the - season a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus acute respiratory disease associated with adenovirus serotype -four states clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) multiplex masstag-pcr for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: a newly emerging respiratory pathogen antigenic and genetic variability of human metapneumoviruses genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness the role of human metapneumovirus in upper respiratory tract infections in children: a -year experience characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia identification of a new human coronavirus human bocavirus detection of human bocavirus in hospitalised children human bocavirus infections in hospitalized children and adults high incidence of human bocavirus infection in children in spain respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants multipathogen infections in hospitalized children with acute respiratory infections sole pathogen in acute bronchiolitis: is there a role for other organisms apart from respiratory syncytial virus? diagnostic system for rapid and sensitive differential detection of pathogens a metagenomic survey of microbes in honey bee colony collapse disorder a new arenavirus in a cluster of fatal transplant-associated diseases the study was supported by the li ka-shing foundation (respari network) for discovery of new emerging viruses, the french agency for the development for surveillance and investigation of epidemic situations in southeast asia (sisea project), the national science and technology major project ( zx - ) of the chinese ministry of health for the establishment of pathogen and immuno-response detection platforms for respiratory and central nervous system viral infections, the areva foundation which supports the salary of dr. wei wang. we thank drs. franç ois freymuth (virology laboratory, chu caen, france) and sylvie van der werf (national reference center for influenza virus in northern france, institut pasteur paris), and dr. jean-claude manuguerra (urgent response to biological threats) for having facilitated this work. we also thank y.m. zheng for manuscript preparation. the authors declare no conflict of interest. key: cord- - cmgu rf authors: mcerlean, p.; shackelton, l.a.; lambert, s.b.; nissen, m.d.; sloots, t.p.; mackay, i.m. title: characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: cmgu rf background: human rhinoviruses (hrvs) are some of the earliest identified and most commonly detected viruses associated with acute respiratory tract infections (artis) and yet the molecular epidemiology and genomic variation of individual serotypes remains undefined. objectives: to molecularly characterise a novel hrv and determine its prevalence and clinical impact on a predominantly paediatric population. study design: nucleotide sequencing was employed to determine the complete hrv-qpm coding sequence. two novel real-time rt-pcr diagnostic assays were designed and employed to retrospectively screen a well-defined population of specimen extracts to identify the prevalence of hrv-qpm during . results: phylogenetic studies of complete coding sequences defined hrv-qpm as a novel member the genus rhinovirus residing within the previously described hrv-a sub-lineage. investigation of the relatively short vp sequence suggest that the virus is resistant to pleconaril, setting it apart from the hrv a species. sixteen additional hrv-qpm strains were detected ( . % of specimens) often as the sole micro-organism present among infants with suspected bronchiolitis. hrv-qpm was also detected in europe during , and a closely related virus circulated in the united states during . conclusions: we present the molecular characterisation and preliminary clinical impact of a newly identified hrv along with sequences representing additional new hrvs. acute respiratory tract infections (artis) are a leading cause of human morbidity worldwide and are most frequently viral in origin. many newly identified viruses have recently been associated with arti, including human metapneumovirus (hmpv; van den hoogen et al., ) , human coronaviruses (hcov) nl (van der hoek et al., ) and hku (woo et al., ) , human bocavirus (hbov; allander et al., ) and the ki and wu polyomaviruses (allander et al., ; gaynor et al., ) . human rhinoviruses (hrvs) are the most common infectious agents associated with artis, comprising the largest genus of human pathogens, rhinovirus, in the family picornaviridae. despite a significant and growing body of evidence to implicate hrvs in more serious clinical outcomes (gern and busse, ; hayden, ) , the presence and role of more than genetically and serologically distinct hrvs in illness is often unexplored. additionally, the viruses themselves are not considered to be individual entities with distinct circulation patterns as other respiratory viruses are. studies have produced unusual hrv-like sequences or made cursory reports of untypeable or variant hrv strains in the united kingdom (mori and clewley, ) , switzerland (deffernez et al., ) , finland (savolainen et al., ) , belgium (loens et al., ) , australia (arden et al., ) and the united states (lamson et al., ) . many of these strains may represent novel hrv serotypes. during an investigation of picornavirus pcr products we identified a cluster of more than nucleotide sequences demonstrating < % identity with any sequence on genbank (arden et al., ) . we proposed that these belonged to a novel genetic sub-lineage, hrv-a . we further investigated one of these putative viruses and herein present the complete polyprotein coding sequence of a novel hrv, hrv-qpm, which was first detected in an infant with bronchiolitis. clinical specimens (n = ) were predominately nasopharyngeal aspirates (npa; %) collected from patients ( . % male) aged between day and years (mean age . years, median age . years) who had presented to queensland hospitals or general practitioners with symptoms of arti during . npas and purified nucleic acid extracts were stored at − • c. extracts were tested by rt-pcr (onestep, qiagen ® ) for hmpv, hrvs and enteroviruses (hevs), all four non-sars hcovs, hbov, respiratory syncytial virus (hrsv), influenza a and b viruses, parainfluenzaviruses and human adenoviruses (arden et al., ) . real-time rt-pcr employed . - . pmol of oligonucleotide. amplicons were purified when necessary and both strands sequenced (big dye v . , applied biosystems). real-time rt-pcr was performed on the rotor-gene tm (corbett research). first strand cdna synthesis (transcriptor rt, roche) using (t) ata or gene specific primers was followed by second strand synthesis and amplification (platinum ® pfx, invitrogen). oligonucleotide sequences are available upon request. human cell lines (hela-ohio, a , mrc- and wi- ) were inoculated with patient respiratory secretions stored at − • c. inoculated cells were rolled at • c, watched daily for cytopathic effect and hrv-qpm rna levels were monitored by real-time rt-pcr. nucleotide or amino acid sequences were aligned with the program muscle (edgar, ) and, where necessary, backtranslated with the program protogene (moretti et al., ) . maximum likelihood trees were constructed using a gtr + i + model of nucleotide substitution in paup* (swofford, ) , with outgroups as indicated. trees were visualized with treeedit (http://evolve.zoo.ox.ac.uk/software.html). the nucleotide distance matrix for neighbour-joining trees was generated using the kimura two-parameter estimation in mega . (kumar et al., ) . nodal confidence values indicate the results of bootstrap resampling (n = ). initial hrv-qpm sequences were used to design new pcr primers permitting determination of the continuous coding sequence from overlapping fragments, bracketed by partial untranslated regions (utrs). additional primers from hrv (loens et al., ) and hev (caro et al., ; oberste et al., ; simmonds and welch, ) studies supplemented the process. to confirm that our primary sequence belonged to a distinct virus and was not the result of mixed template in the patient's specimen, a second round of rt-pcr and sequencing of overlapping fragments was performed using newly designed hrv-qpm-specific primers targeting rna from the original specimen extract. discrepant sequence results were confirmed by a third round of rt-pcr and sequencing. to determine whether hrv-qpm was a distinct pathogen endemically circulating in brisbane, specimen extracts spanning all of were examined (fig. ) . hrv-qpm b rna was detected in . % (n = ) of extracts by real-time rt-pcr and verified using a confirmatory assay targeting the d region. hrv-qpm prevalence peaked in winter but was also detected in spring and summer. no other micro-organisms were detected in . % (n = ) of positives (table ). there were six co-detections with: hcov-nl , hcov-nl and hbov, hbov, hmpv or hcov- e. overall respiratory picornaviruses were the most frequently detected micro-organisms at . % of microbial detections (n = ) followed by hbov ( . %, isolation of hrv-qpm was unsuccessful due to the age of the inoculum or perhaps because the hrv-a viruses are particularly fastidious. clinical information from the -month-old male index case (yielding hrv-qpm strain ) revealed bronchiolitis following a -day history of rhinorrhoea, cough and respiratory distress. there were no fevers vomiting or diarrhoea noted. the infant had an audible wheeze with scattered crackles in both lung fields, right otitis media and pharyngitis. the chest x-ray showed bilateral patchy changes, worse in the right mid zone, consistent with a non-specific viral lower respiratory infection (lrti). the infant responded to nebulised ventolin and oral prednisolone and was discharged after a h admission. clinical details from the other hrv-qpm positive cases demonstrated the following presenting symptoms and signs: rhinorrhoea, fever, cough and wheeze, with diagnoses including asthma and bronchiolitis, consistent with arti. eight ( . %) of positive patients presented with indications of lrti, most commonly presenting as bronchiolitis ( / ), wheezing ( / ) and one case suspected of pertussis (pcr negative) each in the absence of another micro-organism (table ). the only two adult cases had underlying conditions. analysis of the deduced nucleotide sequence suggested hrv-qpm was a putative new member of the genus rhinovirus. the polyprotein coding sequence spanned nt; shorter than all other reported hrvs and hevs (lukashev et al., ; oberste et al., ; genbank #ef ) . predicted protease cleavage sites, similar to other picornaviruses, were also identified with the unconfirmed division of the polyprotein into typical picornavirus structural (vp - ) and non-structural proteins ( a-c and a-d; netpicorna world wide web server, blom et al., ; skern et al., ; stanway et al., ) . a highly conserved translation initiation site (mgaqvs) shared with other hrvs and hevs was also identified. a striking feature of the hrv-qpm genome was its deviation from all other sequenced members of the hrv-a species. members of hrv-a (represented by the complete coding sequences of serotypes b, , , and ) shared . - . % predicted amino acid identity while hrv-qpm, the sole hrv-a coding sequence to date, shared . - . % identity with hrv-a and . % with hrv-b (serotype ). given the extensive recombination observed in other picornaviruses (lukashev et al., ; simmonds and welch, ) , we next investigated the possible role of recombination in the emergence of hrv-qpm. phylogenies of multiple, distinct hrv polyprotein regions gave no indication of recombination involving hrv-qpm (data not shown), similar to recent findings (simmonds and welch, ) . the paucity of full-genome hrv sequences, however, is a limitation in these analyses. a number of further investigations were undertaken to confirm that hrv-qpm was neither a strain of an existing hrv serotype nor an artefact of pcr amplification or sequencing. the complete a, b and d regions (putatively encoding cleavage products vp , vp and vp ) of all hrv-qpm strains ( - ) were sequenced and compared to representative hrvs and hevs (fig. ) . strains exhibited > % nucleotide identity. these analyses confirmed that hrv-qpm exists as a novel genetic sub-lineage within the hrv-a species. data were obtained from the full vp sequences presented in fig. . a minimum to maximum amino acid identity shared between hrv-qpm and the established hrv and hev serotypes. vp is the predominant viral surface protein and the common target both for serotyping and genotyping studies (oberste et al., ) and the development of antiviral agents (ledford et al., (ledford et al., , . complete vp sequences from all known hrv and hev serotypes were available for comparison to the prototype strain of hrv-qpm, . these data further confirmed that hrv-qpm was similar but distinct from other known hevs and hrvs ( fig. a and b and table ) most likely representing a novel serotype within the hrv-a species. importantly, the putative hrv-qpm vp is - amino acids shorter than that of any other hrv serotype because of several deletions. additional in silico studies predicted that infection by hrv-qpm is not likely to respond to treatment with the capsid binding antiviral, pleconaril. to date all other members of the hrv-a species have proven susceptible to this antiviral compound. two amino acids reportedly conserved among all hrv-b viruses (ledford et al., ) , were altered in the hrv-qpm vp sequence (fig. ) . using the amino acid numbering system from our study, the hrv-qpm-specific changes were tyr to phe and a hydrophilic thr in place of a hydrophobic val or leu . changes at position and are implicated in complete resistance to the antiviral among naturally occurring hrv-b species (ledford et al., ) . a review of sequences located on genbank and in the literature reveals that variants and incompletely characterised hrvs have been noted over many years. most pcr-based hrv studies have targeted the -utr region for nucleotide sequencing of these variants but this region's use as a discriminator of serotype is hindered by the paucity of complete utr sequences available both from known serotypes and hrv variants. the sequences encoding vp /vp and vp have proven more reliable for genotyping and are comparable to serotyping (laine et al., ; ledford et al., ) . a recent study found sequences in belgium (sampled in - ) which appear to be related to hrv-qpm in the vp region (loens et al., ) . in collaboration with other investigators in western europe, we searched small numbers of specimens for hrv- fig. . alignment of predicted vp amino acid sequences hrv-qpm and hrv-a (hrv- a, , and ) and hrv-b (hrv- , , and ) species. numbering based on the hrv-qpm sequence (ef ). the residues of the pleconaril binding pocket are arrowed (majority hrv-b amino acid sequence listed below each arrow, ledford et al., ) . two regions, indicated by overhead lines, appeared to be absent in hrv-qpm when compared with all other hrv vp sequences on genbank (representative data shown here). two residues predicted by ledford to convey complete antiviral resistance to natural hrv-b strains (e.g. in hrv- and ) are indicated by stars. residues identical to hrv-qpm are indicated by dots. the absence of a residue is indicated by a dash. qpm and were able to identify the virus in one country, with a similar prevalence, during its peak respiratory virus season (data not shown). we were unable to find hrv-qpm in the netherlands (specimens from / and / ) in new south wales, australia ( ) or in new zealand ( . however, a very similar virus was circulating in new york during (fig. ) . together, these results suggest that hrv-qpm is not a geographically isolated hrv but one circulating globally. in this study, we have described the detection and distinguishing features of a newly identified hrv together with a summary of its clinical impact on a predominantly paediatric population. hrv-qpm was identified by intensively investigating of more than picornavirus-like sequences obtained during a previous study. in the present study we tested a well characterised set of respiratory specimens, mainly from children, and showed the virus to be present in . % of them, with monthly detection frequencies as high as . % in august and . % in february. hrv-qpm was associated with a variety of clinical syndromes involving mild to severe illness, including a strong linkage with bronchiolitis in the absence of other detectable pathogens. our findings add to recent data about the largely unrecognised diversity of hrv and their importance in clinical illness, in particular, lrti. we found that approximately half of all hrv-qpm detections were from patients with lrt symptoms in whom hrv-qpm was the sole micro-organism detected, suggesting that this hrv at least is associated with more serious illness. detailed prospective clinical studies will be required to compare the prevalence of hrv-qpm in other ill populations and in suitably matched control groups without symptoms. detections of hrv-qpm in europe and a closely related virus in new york suggest that hrv-qpm is not a geographically isolated hrv but a global respiratory pathogen. a small, preliminary study of local specimen extracts collected during other years did not find any additional hrv-qpm strains. this is the first molecular epidemiology study to address the annual prevalence of an individual hrv and these findings may be suggestive of the nature of all hrv serotypes. immunity resulting from infection may protect a particular community from a certain serotype until a suitably large naïve community, either due to increasing numbers of non-immune infants and young children or adults with waning immunity, is available to again facilitate that serotype's unfettered infection and transmission. this period may encompass several years. it will be important to carry out future 'hrv-hunting' studies over many respiratory seasons to maximise the number of new viruses detected, overcome the restricting effects of herd immunity and better identify the length of time between cycles of infection for each hrv. since their discovery in the s, infection by hrvs has been synonymous with the 'common cold' and, despite reports of association with more serious clinical outcomes (gern and busse, ; hayden, ; kaiser et al., ) , these viruses are still considered to be of minor clinical significance by many. consequently, the genus rhinovirus has been poorly characterised by modern standards; only six unique genomes from over hrv serotypes currently reside on genbank. the paucity of sequence data undoubtedly parallels our understanding of individual serotypes. in a time when molecular diagnostics are considered the gold standard, a lack of sequence reduces our detection capabilities and therefore limits our understanding of the seasonal distribution and recurrence of each serotype. in turn, this has perpetuated the concept that the genus is a collection of "just rhinoviruses" rather than a large number of individual viruses each worthy of detailed characterisation. detailed studies of hrv-qpm and related hrv-a s may now determine if this novel group represents a recently emerged global subset of hrvs that are more frequently associated with clinically severe respiratory outcomes or a previously unidentified viral group that has been circulating for many years. these and future findings will hopefully invigorate research into the genomics and epidemiology of these long ignored and clinically important respiratory viruses. identification of a third human polyomavirus cloning of a human parvovirus by molecular screening of respiratory tract samples frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections cleavage site analysis in picornaviral polyproteins: discovering cellular targets by neural networks molecular strategy for 'serotyping' of human enteroviruses amplicon sequencing and improved detection of human rhinovirus in respiratory samples identification of a novel human polyomavirus from patients with acute respiratory tract infections association of rhinovirus infections with asthma rhinovirus and the lower respiratory tract chronic rhinoviral infection in lung transplant recipients mega : integrated software for molecular evolutionary genetic analysis and sequence alignment phylogenetic analysis of human rhinovirus capsid protein vp and a protease coding sequences confirms shared genus-like relationships with human enteroviruses masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during insights into the genetic basis for natural phenotypic resistance of human rhinoviruses to pleconaril vp sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season recombination in circulating human enterovirus b: independent evolution of structural and non-structural genome regions protogene: turning amino acid alignments into bona fide cds nucleotide alignments polymerase chain reaction and sequencing for typing rhinovirus rna molecular evolution of the human enteroviruses: correlation of serotype with vp sequence and application to picornavirus classification enterovirus is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus frequency and dynamics of recombination within different species of human enteroviruses human rhinovirus : complete nucleotide sequence and proteolytic processing signals in the capsid protein region the complete nucleotide sequence of a common cold virus: human rhinovirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia this work was supported by nhmrc project grant and the royal children's hospital foundation grants - and - sponsored by the woolworth's fresh futures programme. we are grateful to dr. lia van der hoek and dr. eric c.j. claas (the netherlands), dr. william rawlinson (nsw, australia) and dr. lance jennings (new zealand) for local hrv-qpm testing. we thank queensland heath pathology service for the provision of clinical specimens. key: cord- - iqpl p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: - - journal: viral infections of humans doi: . / - - - - _ sha: doc_id: cord_uid: iqpl p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under years and occur in all seasons [ , ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ ] and with signifi cant direct and indirect healthcare expenditure [ , ] . ari incidence is highest in the fi rst years of life, with up to episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ , - ] . in preschool-aged children, nearly % of general practitioner visits are for ari [ ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ , ] , and smooth muscle cells [ ] but not in monocytes [ ] or dendritic cells (dcs) [ ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. . ). furthermore, many modern studies ( ) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and ( ) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ - ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ , ] . in the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ , ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ ] . the ccu functioned for years ( - ) , and it was here in that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. . ) [ , ] . propagation failed once this tissue was expended [ , ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ - ] . in renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in [ - ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in , few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ ] . later, price ( ; the jh strain) and then pelon and co-workers ( ; , strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ , ] . the early hrvs were initially classifi ed as echoviruses (echo ; later hrv- ) [ ] . at the same time, propagation of the hgp (hrv- ) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ , ] . despite the challenges [ ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from to tcid /ml [ - ] and growth differences among the different virus types. additionally, cell age after plating (< h), inoculum volume (relevant to the culture vessel), medium ph ( . - . ), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ - ] . the hrvs can grow at temperatures above °c (some prefer that under certain conditions) [ ] , but rolling at °c, preceded by a - -h stationary incubation period [ ] , has historically provided the highest yield and fastest in vitro hrv growth [ , , , ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ , ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in [ ] . the fi rst classical strains were officially named in [ ] , the last in [ ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ , - ] . in hrv-c and w (another hrv-c) were shown to grow using organ culture [ ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc ) [ ] . isolation identifi ed hrvs in ~ % of adults with aris, associated with . illnesses per year [ ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ - ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ - ] . with reliable detection levels that extend from as few as tcid /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ , ] . the common understanding of the systemic [ - ] or symptomatic [ , ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ ] . the ′ untranslated region (utr; figs. . and . ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in [ ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ , , , , - , , - ] . the ′utr is comprised of a number of conserved sequence "islands" (fig. . ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ , ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ ] . the pcr primers of broadly reactive conventional rt-pcr [ , ] and rt-rtpcr [ , ] assays are shown these primers or changing the method of employing them [ - ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ ] , and by comparison of multiple primer sets using the same specimens [ ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying -fold fewer tcid /ml or fold fewer genome copies than agarose gel detection of amplicon [ , , ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ , , - ] , and some have gone on to be approved for clinical laboratory use [ ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd - per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ , ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp , ′utr+vp +vp (called hereafter vp /vp ), or ′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ ] . the vp /vp region (fig. . ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~ hrvs from a challenging biological substrate, clinical specimens [ ] . screening of airway specimens for hrvs is not routine [ ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. . ) [ ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells h after infection [ , ] . single-stranded infectious rna forms after ris start to accumulate [ ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ ] . virions are synthesized from to h after infection and reach maximum release levels at - h [ ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease a ( a pro ) of hrv-b may directly cleave eukaryotic initiation factor g (eif g) when bound to eif e [ , ] . the eifs have key roles in initiation and rate control of host cell translation [ ] . host cellular protein production is virtually replaced by hrv-b proteins after only h of infection [ ] . hrv-b -infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf- , stat , nf-κb) and shutting down host cell protein synthesis. protease c ( c pro ) from hrv-a targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ , ] . recombinant a pro protein from hrv-a ,hrv-a , hrv-b , hrv-b , hrv-c , and hrv-c exhibited differing specifi cities and kinetics against eif g as well as npc components demonstrating functional diversity between hrv types [ ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately kb positive sense rna genome (fig. . ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by a pro and c pro into the proteins ( p -p ) and structural peptides ( vp -vp ; vp and vp derive from the vp precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. . ). there are complete hrv polyproteins on the genbank database ( fig. . ). the fi rst complete hrv genome sequence (hrv-b ) was described in [ ] followed by hrv-a in [ ] and hrv-a b in [ ] (fig. [ ] . sequencing of the vp /vp region was completed for all classical strains in [ ] , and the complete set of d regions were available in [ ] . currently there are at least named hrv-c vp regions the current spectrum of complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v . [ ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version (poisson model, bootstraps with consensus support shown at the nodes where space permitted [ ] ) (reprinted with permission from miller and mackay [ ]) available and complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many ′utr and vp /vp sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of ′utr, vp , and vp /vp subgenomic regions for useful representation of hrv species and types [ , ] . recombination, the process of genetic exchange which results in a chimeric genome [ ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ ] have been made based on examination of the available set of hrv coding and noncoding regions [ ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ , , ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ , , , ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the ′utr or at the ′utr/vp junction [ , , ] but rarely in coding sequence ( a [ ] or c [ ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the - nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a a, hrv-a , hrv-b , hrv-b , and hrv-a ), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b , was described in [ ] followed by hrv-a a in [ ] , hrv-a in [ ] , hrv-a in [ ] , and hrv-a in [ ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. . ) [ ] . the hrv capsid shell is composed of protomers, each comprising one copy of the viral proteins vp , vp , vp , and vp . vp , vp , and vp (each ~ kda) are to some extent exposed on the capsid surface, whereas vp (~ kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp , vp , and vp are each formed by a convoluted set of protein sheets and loops [ ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b and hrv-a : nim- a (located in vp ), nim- b (vp ), nim-ii (vp and vp ), and nim-iii (vp and vp ) [ ] . antigenic sites identifi ed on hrv-a are called a, b, and c [ ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. . ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of > viral types (table . ). this accounts for the combination of hrv-a a and -a b, exclusion of hrv- , which is actually ev-d despite confusion over acid liability [ - ] and combination of hrv-hanks which is actually hrv-a [ ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ ] . species within the genus share > % amino acid (aa) identity in the polyprotein and in c+ cd and > % aa identity in p ( fig. . ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than . %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ ] . a variant of the same hrv type shares - % aa identity or more in vp [ ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least % different in its amino acid identity from any other genus. in a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in , and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c , formerly nat ; hrv-c , f. nat ; hrv-c , f. qpm; hrv-c , f. c , etc.; table . ) [ ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp sequence or provisionally ("c_pat," table . ) based on vp /vp [ ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the ′utr can be and still is used [ , ] for hrv genotyping, but it is a more problematic region than vp or vp /vp because of the recombination activity that affects this region, especially among the hrv-cs [ ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥ % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ ] . there are currently types within hrv-c (which includes the types once grouped together under hrv-"a ," hrv-x, and hrv-ny clades), hrv-a types, and hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c versus hrv-a simplot data projected onto the hrv-c pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a , gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ ] . attachment of the vldl-r involves adjacent vp molecules. magnifi ed vp area represents one half of a vldl-r footprint [ ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature ( - °c versus °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ , - ] . these two groups correlate with receptor usage [ ] (table . ) and possibly with the titer of the inoculum employed [ ] . in it was proposed to abandon this terminology in favor of a sequential numbering system [ ] . picornaviruses recognize a variety of cellular receptors [ , , ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ , ] . the capsid of the majority of classical hrvs ( n = ) [ ] interacts with the amino-terminal domain of the kda intercellular adhesion molecule (icam- ; cd ) [ - ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ , , ] . icam- interacts with its receptor, leukocyte function antigen- (lfa- ), and plays a role in recruitment and migration of immune effector cells [ ] . the minor group [ ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ , , ] . in andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ , ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ ] . when sequences from other subgenomic regions, including p , c, and cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table . ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ , ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ , ] . hrv-a and hrv-a are also likely the same serotype [ ] . a full list of genetically close serotype pairings was presented by ledford et al. [ ] hrv-c nomenclature was defi ned in and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp /vp data can be confi rmed with vp sequence and the provisional number removed (e.g., c_pat to c_pat have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ , , , - ] . for the hrv-cs, fi rst described as the "hrv-a " clade (not to be confused with the single virus, hrv-a , this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in [ ] , sequencing of ′utr and vp /vp has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. . ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ ] ; hence, prior to , detection of the full spectrum of ≥ hrvs did not occur. after , the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over months examined hrv frequency and diversity in specimens from healthy children ( - years of age) [ ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between and distinct hrvs at a single location [ , , ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ , , ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ ] . in populations of mixed age, the majority of hrv detections occur in children [ ] . among specimens from healthy children, over a third ( %) were hrv positive. children less than years of age ( % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than years old ( % hrv positive) [ ] . healthy adults in the military [ , ] , at university [ ] , at home [ - ] , and in the workplace [ ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ , ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ ] including those with febrile illnesses [ ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as - years of age) with % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. . ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. . ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ , , ] (and, peculiarly, on a monday [ ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ ] and hrsv [ ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" - days after return to school from holidays, in a range of climates [ - ] . this was particularly obvious among asthma hospitalizations of children ( - years of age) in ontario, canada, which peaked at weeks - across a decade [ ] . upon investigation, hrvs were the most prevalent of the viruses found in a -year analysis of emergency room presentations in ontario [ ] . hrvs also predominate during "hay fever season" [ ] . although a defi ned seasonality is not always found in the tropics [ ] , this may sometimes be due to testing that does not include hrvs [ , ] or only some hrvs [ ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during - months, or more types can co-circulate [ , ] [ ], dropping [ , ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for . % of any virus detected in a birth cohort followed for months [ ] and, in another cohort, occurred twice in two children, within a -month period [ ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ , , , - ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ , - ] or have no impact on the clinical outcome in their host [ - ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet % or more of hrsv, hmpv, ev, and ifv detections and % of hcov-nl detections can be found in the company of another virus [ ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ , ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ , ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ , ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for days postvaccination [ ] . this study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by days or more, during times of a prolonged epidemic [ ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by % overall [ ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ ] . the mechanism underneath these opposing outcomes is unclear. during the heyday ( s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv- [ , ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to respiratory viruses (fig. . ) [ , ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ . ). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ ] . during an -week period that spanned peak h n pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as h n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ ] . reports of subjects with continuous and extended (greater than - weeks) periods of hrv positivity [ , ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ ] . hrv rna has been detected days prior to symptoms commencing and for as long as or more weeks after they cease [ , - ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ ] will not detect overlapping serial infections (fig. . ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ ] . hrv shedding normally ceases within - days, after signs and symptoms have stopped [ , , , , , ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ , , , ] . few studies [ ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ , ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of versus days [ ] . multiple detection of the same hrv type ( % identical hrv- a sequence in each patient over time) extended to months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ , , ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ , ] . some studies employ sensitive symptom scoring systems [ ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ ] . infants frequently exposed to other children have more asymptomatic viral infections [ ] . among infected adult family members, asymptomatic infections are more likely [ ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ ] . it is clear that a proportion, around fi ve to % of study-defi ned asymptomatic control populations [ , , ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ , ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ , , ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ , , ] . a birth cohort of children enrolled and sampled when ill and every months for months identifi ed hrvs - % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ ] . the childhood origins of asthma (coast) birth cohort followed infants at high risk for allergies and asthma for months and identifi ed hrv infections as preceding (mean age of fi rst detection, months) those of hrsv (mean age at least months), and hrvs were found in % of asymptomatic versus % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ ] . in a study of children with asthma sampled weekly for weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ ] . a convenience population of healthy children ( - years old) without asthma were followed during at least three seasons, and picornaviruses were detected in % of specimens ( % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = , for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( - ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although of the infections came from households with an infected sibling [ ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ , ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ , ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ ] . an hrv was once isolated from feces [ ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ ] , with gastroenteritis [ ] , and in a child with pericarditis [ ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ ] , regardless of inoculation route [ ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ , ] . the incubation period between infection and onset of virus shedding into nasal secretions is - days with shed viral titers peaking in adults between days and [ , ] . the time until successful hrv transmission among adults in a childless family setting is usually - days and requires the donor to be shedding at least tcid at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ ] . the lungs have been shown to host replicating hrv [ ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of . ml to ml of suspension [ - , , - ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ , ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, % (donor to objects to recipient) and % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ ] . even under observation, eye rubbing ( . h − - . h − ) and nosepicking ( . h − - . h − ) occur frequently [ , ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ , ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ ] despite viral loads in nasal washings peaking at . × tcid /ml [ ] . experimental transmission was also reportedly ineffi cient [ ] . in contrast, "naturally" close-quartered military populations, interacting over - weeks, experienced rapid spread of hrvs to > % of the group [ ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches - % [ , ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva ( % compared to % of hand washes and % of nasal swabs) [ , , ] and from only . % of participants exposed to large-particle aerosols [ ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ , ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ , ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ ] . when exposed to liters of a small-particle aerosol, tcid of hrv- was associated with fever and prominent tracheobronchitis in antibody-free (< : ) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ , ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from % of ~ -hourold fi lters placed to sample air in offi ce buildings [ ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than days after they were removed for servicing [ ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ , , ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. . ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ ] and do so despite not hosting hrv replication directly [ ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ ] . the type iii ifns (ifn-λ or il- , ifn-λ or il- a, and ifn-λ or il- b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ , ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), ′ ′oas/rnasel, and the mx proteins [ ] . ifn-α upregulates expression of mxa, ′ ′-oas, and pkr [ ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, ′ ′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene (mda , specifi c for picornaviruses and others) [ , ] . these rna helicases recognize either rna with a ′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ , ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a [ ] , ifn regulatory factor (irf)- homodimerization is interfered with hrv-b which limits ifn-β induction [ , ] , and mda is degraded by hrv-a a but not hrv-a [ ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr , tlr , tlr , and tlr recognize nucleic acids and are also involved in innate antiviral responses. tlr and tlr identify g/u-rich ssrna from endocytosed viruses, while tlr recognizes unmethylated cpg dna present in dna viruses [ , ] . tlr and tlr are found on the cell surface and recognize hrv or hrsv proteins, respectively [ , ] , and tlr recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ ] . tlr activation can reduce ′ ′oas and mxa mrna expression and ip protein in adolescents with asthma compared to healthy controls [ ] . tlr activation did not result in a similar disparity [ ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table . ) [ , , ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h - -like and t h- -like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h - cellular response is important in managing cellular immunity and producing interleukin (il)- and ifn-γ. the t h - cellular response manages humoral immunity and stimulates b cells via il (initiating production of ige), il (infl uencing eosinophils), and il (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ ] . t h- lymphocytes, opposing t h- lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ ] . the t h- response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h- state in mice and probably in humans [ ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h - response is comparatively defi cient in people with asthma [ , ] . this is problematic as an increased t h - -like cytokine response, deduced from higher sputum mrna ifn-γ/il values, speeds clearance of hrv and symptom amelioration [ ] . one possible cause of the t h - defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ ] . the "hygiene hypothesis" [ , ] posits a pathway for an asthma etiology described [ ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h- -like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a a infection revealed some signifi cant differences that were found between cell types and response to infection [ ] . these included immune response genes (il b, il , il , il f , il ) and airway remodeling genes (loxl , mmp , fn ) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ , - ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b was found to promote release of il from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il (drives t h - development) [ ] . however, neither il nor il was signifi cantly induced in asthmatic adult volunteers in response to hrv-a compared to healthy subjects [ ] . while ifn-α was detected after transfection of dcs with hrv-b ssrna, low tnf-α and il levels were also noted [ ] . it was posited that the reduced il could indicate negatively affected local immunity possibly predisposing to secondary infections [ ] . infection of stromal lung cells by hrv-b triggered exaggerated levels of the pleiotropic il (an il type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a . g-csf and il (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ , , ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam- than those of nonatopic adults [ ] as do normal subjects infected by the major group hrv-b [ ] . by contrast, ifn-γ and il , which appear later postinfection, downregulate icam- expression in infected cells [ ] and encourage infi ltration of neutrophils [ ] , respectively. changes in icam- levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il- β proinfl ammatory properties; enhances adhesion molecule expression including icam- ; induces il- receptor gm-csf a granulocyte and monocyte growth factor il- stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il- t h differentiation, promotes ige synthesis il- activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il- /cxcl- neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il- anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il- , il- , and tnf-α irf a master hub, regulating antiviral immunity ip /cxcl chemoattractant for activated t h and nk cells tnf-α proinfl ammatory activity similar to il- β; activates neutrophils; induces vascular permeability mpc- a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp an il- -like cytokine that activates myeloid dcs to induce naive t cells into t h cells producing il- , il- , and tnf-α; induced by hrv in the presence of il- bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ , ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least days to weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least weeks [ , - ] , falling faster than serum levels [ , ] . there is also some evidence for a degree of nasal immune memory [ ] . volunteers with pre-study serum antibody could still be infected in some studies [ , , ] , but not in others [ ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ , ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ , ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥ : existed; as levels grew from , so did levels of resistance to infection [ , ] . adults were protected by serum titers of : - : [ , ] . the trend was interrupted by adults in the - year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ , ] . however, if it were possible to identify "master" strains [ ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ , ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ , , , ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ , ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a and hrv-a [ ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a sera had a higher titer for hrv-a than anti-hrv-a sera did for hrv-a [ ] . over strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a and hrv-a are linked via hrv-a , hrv-a , and hrv-a (anti-a serum neutralizes hrv-a , anti-a neutralizes hrv-a , and anti-a neutralizes both hrv-a and hrva-a [ , ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with to > % of colds positive for an hrv [ , , ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the h n pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ , , ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ , ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ , ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day and was more persistent up to days later [ , ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ , ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ , ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ , ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ , ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h type response; and (iv) hrvs may simply exacerbate that which already exists [ ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ , , ] , or a mix of both. in children with asthma, viruses have been detected in at least % of exacerbations ( % picornaviruses, probably hrvs [ ] ) and in % of adults [ ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ , - ] . bacteria are not major factors in wheezing exacerbations [ ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ , , ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ , ] . hrvs even predominated in some culture-based studies of wheeze [ , ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ , , ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th -type t cells (th cells) [ ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il is signifi cantly higher in serum and nasal aspirates than for hrsv [ ] . ip was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ , , , ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ , , , - ] although, in some instances, an exacerbation is the only sign of infection [ ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ ] . bacteria were often present with ifv, but not with hrvs [ ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ ] although hospitalizations may be fewer for hrv-cs than the other species [ ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ , ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ - ] . since a longitudinal day-care study in , the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ , ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction ( %) due to the target bacteria [ ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ , , ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ , ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ , ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of - % of children with aom [ , ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ ] . picornaviruses have been detected in % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from % of children with recurrent aom and/or adenoid hyperplasia [ , ] . in a cohort of children followed from to months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ ] . viruses, most often hrvs ( . % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes ( % of aom without ari) [ ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between and months of age ]. hrvs accounted for % of viruses detected and % of specimens with a single virus detected. this dominance was maintained even through the h n infl uenza pandemic [ ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ ] . the presence of hrv-b was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ ] . hrv infections result in more copd exacerbations (~ % of cases [ ] ) than any other virus identifi ed to date [ , ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ ] . adding the measurement of an infl ammatory marker in the serum, like il- , further improves the speed of predicting an infectious etiology for exacerbations of copd [ ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ , ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of years [ ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ , ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ , ] . infections causing pneumonia vary with age and vaccination status [ ] . viruses can be detected in up to % of infants ( - months of age) with pneumonia, and these cases follow a seasonal pattern [ , ] . bacteria can also be detected in over % of infants and older children, the elderly, and those with severe cap [ , ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising - % of childhood pneumonia cases [ , ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across or more years, which presumably would account for seasonal variation in virus prevalence [ , , , ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ , , , ] . there are laboratory data which support this [ ] as well as observational data showing a high proportion of hrvbacterial co-detections [ , ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ , , , ] . viral-bacterial coinfections can comprise % of patients, while viral-viral ( - %) and bacterial-bacterial ( - %) are much less common [ , , , , ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ , , ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ ] . acute bronchitis (less than -week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ , ] . despite testing, a third of cases remain without a viral etiology [ ] . tracheobronchitis resulted from some hrv-a infection of volunteers [ ] . chest pain and cough have been reported in half or more of adults with hrv infection [ ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ , ] . bronchiolitis occurs seasonally, especially in winter, in infants ( - months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ , ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a -year prospective cohort of very low birth weight infants in buenos aires, argentina [ ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ , ] . the precise role for viruses and bacteria in sinusitis is still unclear [ ] . sinusitis is a common comorbidity in those with asthma [ ] . the in situ presence of hrv-b rna in maxillary sinus epithelium was reported in seven of adults with acute sinusitis [ ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ , ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a [ ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ , - ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ , ] , albeit in very low numbers by nonmolecular studies [ ] and without a significantly different clinical outcome from non-hrv aris in these patients [ ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ , , ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ , ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ ] despite good results in experimental tests [ ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ - ] . however, continual hand washing with extra ingredients resulted in skin irritation [ ] . the experimental testing [ , ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ ] . anticholinergic agents have proven useful to reduce rhinorrhea [ ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ ] . when initiated early in the illness, a combination of antiviral (ifn-α b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ ] . antiviral agents (table . ) require early application to effectively precede the pathogenic immune response to hrv infection [ ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. . ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented in hrv detections. we do not yet know their niche or clinical impact. it may be possible that hrv-bs are the most well adapted of the hrvs, causing little to no detectable clinical impact, or they may create a different impact than that which we expect, or they may be a species in decline. the jury remains out on whether hrvs cause or are involved in the development of asthma or merely trigger exacerbations once asthma is established. with a very high healthcare impact from asthma around the world and atopic conditions that may be exacerbated by hrvs on the rise, this is an important area for further investigations. picornavirus infections: a primer for the practitioner who estimates of the causes of death in children picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season cost burden of viral respiratory infections: issues for formulary decision makers the cost of communitymanaged viral respiratory illnesses in a cohort of healthy preschoolaged children epidemiology of viral respiratory infections serial viral infections in infants with recurrent respiratory illnesses community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a -month period morbidity and treatment in general practice in australia incidence of acute otitis media and sinusitis complicating upper respiratory tract infection: the effect of age rhinovirus replication causes rantes production in primary bronchial epithelial cells rhinoviruses infect the lower airways mechanism of rhinovirusinduced changes in airway smooth muscle responsiveness rhinovirus enters but does not replicate inside monocytes or airway macrophages the ssrna genome of human rhinovirus induces a type i ifn response but fails to induce maturation in human monocyte-derived dendritic cells observations on the incidence and distribution of the common cold in a rural community during and transmission of the common cold to volunteers under controlled conditions. iv. specifi c immunity to the common cold the common cold a short history of the common cold the medical features of the papyrus ebers the common cold cold wars: the fi ght against the common cold in pursuit of the common cold. london: william heinemann medical books limited propagation of common-cold virus in tissue cultures the cultivation in human-embryo cells of a virus (d.c.) causing colds in man rhinoviruses: basis for a numbering system. i. hela cells for propagation and serologic procedures rhinoviruses: basis for a numbering system. ii serologic characterization of prototype strains a collaborative report: rhinoviruses-extension of the numbering system a collaborative report: rhinoviruses -extension of the numbering system from to frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections clinical features and complete genome characterization of a distinct human rhinovirus genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) ratifi cation vote on taxonomic proposals to the international committee on taxonomy of viruses ratifi cation vote on taxonomic proposals to the international committee on taxonomy of viruses rhinoviruses and common colds a cytopathogenic agent isolated from naval recruits with mild respiratory illnesses the isolation of a new virus associated with respiratory clinical disease in humans classifi cation of the " " virus as echo and further study of its properties some virus isolations from common colds. i. experiments employing human volunteers a plaque method for assaying some viruses isolated from common colds problems in characterizing and identifying an apparently new virus found in association with mild respiratory disease in recruits relation between naturally acquired immunity and infectivity of two rhinoviruses in volunteers quantitative rhinovirus shedding patterns in volunteers short-duration exposure and the transmission of rhinoviral colds production of tracheobronchitis in volunteers with rhinovirus in a small-particle aerosol transmission of rhinovirus colds by self-inoculation reproducible plaquing system for rhinovirus serotypes in hela cells -agarose suspension micro-neutralization test for identifi cation of rhinovirus serotypes growth, plaque production and cationic stabilization of rhinovirus type (echovirus ) enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium an rna replication-center assay for high content image-based quantifi cations of human rhinovirus and coxsackievirus infections rhinoviruses replicate effectively at lower airway temperatures epidemiology of the common cold in military recruits with emphasis on infections by rhinovirus types a, , and two unclassifi ed rhinoviruses viruses as precipitants of asthma symptoms iii. rhinoviruses: molecular biology and prospects for future intervention characterization and classifi cation of echo -rhinovirus-coryzavirus agents rhinoviruses: a numbering system immunofl uorescence versus xtag multiplex pcr for the detection of respiratory picornavirus infections in children characterisation of a newly identifi ed human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis a novel group of rhinoviruses is associated with asthma hospitalizations rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illness in infants molecular modeling, organ culture and reverse genetics for a newly identifi ed human rhinovirus c rhinovirus infections in an industrial population i. the occurrence of illness detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr epidemiology of documented viral respiratory infections and acute otitis media in a cohort of children followed from two to twenty-four months of age nested pcr for specifi c detection and rapid identifi cation of human picornaviruses improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay frequency and natural history of rhinovirus infections in adults during autumn a recently identifi ed rhinovirus genotype is associated with severe respiratory-tract infection in children in germany single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response communityacquired pathogens associated with prolonged coughing in children: a prospective cohort study a communitybased, time-matched, case-control study of respiratory viruses and exacerbations of copd detection of human rhinovirus rna in nasal washings by pcr real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses rhinovirus viremia in children with respiratory infections high detection frequency and viral loads of human rhinovirus species a to c in fecal samples; diagnostic and clinical implications metagenomic analyses of viruses in stool samples from children with acute fl accid paralysis use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms rhinovirus/enterovirus rna in tonsillar tissue of children with tonsillar disease quantifi cation of microorganisms: not human, not simple, not quick polymerase chain reaction amplifi cation of rhinovirus nucleic acids from clinical material new respiratory enterovirus and recombinant rhinoviruses among circulating strains typing of human rhinoviruses based on sequence variations in the ′ non-coding region relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection incidence, etiology, and symptomatology of upper respiratory illness in elite athletes environmental contamination with rhinovirus and transfer to fi ngers of healthy individuals by daily life activity prevalence of viral respiratory tract infections in children with asthma rapid detection of human rhinoviruses in nasopharyngeal aspirates by a microwell reverse transcription-pcr-hybridization assay human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms role of respiratory viruses in acute upper and lower respiratory tract illness in the fi rst year of life respiratory viruses, symptoms, and infl ammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease rhinovirus identifi cation by bgl i digestion of picornavirus rt-pcr amplicons detection of rhinovirus, respiratory syncytial virus and coronavirus infections in acute otitis media by reverse-transcriptase polymerase chain reaction effi cacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of double-blind, randomized, placebo-controlled trials comparison of results of detection of rhinovirus by pcr and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested pcr polymerase chain reaction for human picornaviruses detection of respiratory syncytial virus, parainfl uenzavirus , adenovirus and rhinovirus sequences in respiratory tract of infants by polymerase chain reaction and hybridization detection of enteroviruses and rhinoviruses in clinical specimens by pcr and liquid-phase hybridization virological and serological analysis of rhinovirus infections during the fi rst two years of life in a cohort of children simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays acute, chronic and persistent enterovirus and poliovirus infections: detection of viral genome by seminested pcr amplifi cation in culture-negative samples multicenter evaluation of a commercially available pcr assay for diagnosing enterovirus infection in a panel of cerebrospinal fl uid specimens improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr effects of upper respiratory tract infections in patients with cystic fi brosis amplicon sequencing and improved detection of human rhinovirus in respiratory samples respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children detection of respiratory viruses with two-set multiplex reverse transcriptase-pcr assay using a dual priming oligonucleotide system usefulness of published pcr primers in detecting human rhinovirus infection differential detection of rhinoviruses and enteroviruses rna sequences associated with classical immunofl uorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis amplifi cation of rhinovirus specifi c nucleic acids from clinical samples using the polymerase chain reaction newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses multicode-plx system for multiplexed detection of seventeen respiratory viruses diagnostic system for rapid and sensitive differential detection of pathogens masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused infl uenza-like illness in new york state during different cytokine profi le and eosinophil activation are involved in rhinovirus-and rs virus-induced acute exacerbation of childhood wheezing development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fl uid microbead-based assay comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder assay and respifinder smart assay diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort molecular diagnosis of respiratory virus infections microarray-based detection and genotyping of viral pathogens characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing rapid multiserotype detection of human rhinoviruses on optically coated silicon surfaces rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array sers substrate proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types prospective genotyping of human rhinoviruses in children and adults during the winter of - differential processing of nuclear pore complex proteins by rhinovirus a proteases from different species and serotypes replication of rhinovirus rna the structure and replication of rhinoviruses the eif g-eif e complex is the target for direct cleavage by the rhinovirus a proteinase eukaryotic initiation factor gii (eif gii), but not eif gi, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection human rhinovirus infection of hela cells results in the proteolytic cleavage of the p cap-binding complex subunit and inactivates globin mrna translation in vitro inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus rhinovirus c protease can localize in the nucleus and alter active and passive nucleocytoplasmic transport upper respiratory tract viral infection and mucociliary clearance els the complete nucleotide sequence of coxsackievirus a systematic nomenclature of picornavirus proteins the complete nucleotide sequence of a common cold virus: human rhinovirus human rhinovirus : complete nucleotide sequence and proteolytic processing signals in the capsid protein region the nucleotide sequence of human rhinovirus b: molecular relationships within the rhinovirus genus genome-wide diversity and selective pressure in the human rhinovirus new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus vp sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds ref type: computer program) mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods from sneeze to wheeze: what we know about rhinovirus cs recombination in the evolution of picornaviruses molecular characterization of human rhinovirus fi eld strains isolated during surveillance of enteroviruses phylogenetic relationships and molecular adaptation dynamics of human rhinoviruses recombination and selection in the evolution of picornaviruses and other mammalian positive-stranded rna viruses recombination of poliovirus rna proceeds in mixed replication complexes originating from distinct replication start sites the mechanism of rna recombination in poliovirus sequencing and analyses of all known human rhinovirus genomes reveals structure and evolution analysis of genetic diversity and sites of recombination in human rhinovirus species c structure of a human common cold virus and functional relationship to other picornaviruses crystal structure of human rhinovirus serotype a (hrv a) the structure of human rhinovirus human rhinovirus at . a resolution crystallization and preliminary x-ray analysis of human rhinovirus serotype (hrv ) picornavirus-receptor interactions alignment of capsid protein vp sequences of all human rhinovirus prototype strains: conserved motifs and functional domains full-length human immunodefi ciency virus type genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination members of the low density lipoprotein receptor family mediate cell entry of a minor group common cold virus virus taxonomy: eighth report of the international committee in taxonomy of viruses the major and minor group receptor families contain all but one human rhinovirus serotype a probable new human picornavirus associated with respiratory diseases human rhinovirus identifi ed as human enterovirus by vp -based molecular diagnosis classifi cation and nomenclature of viruses: ninth report of the international committee for the taxonomy of viruses clinical characteristics and genetic variability of human rhinovirus in mexico prevalence of and risk factors for human rhinovirus infection in health aboriginal and non-aboriginal western australian children ′ noncoding region alone does not unequivocally determine genetic type of human rhinovirus strains some further virus isolations from common colds serotypes of viruses (rhinoviruses) isolated from common colds demonstration of dual rhinovirus infection in humans by isolation of different serotypes in human heteroploid (hela) and human diploid fi broblast cell cultures relationship of rhinovirus infection to mild upper respiratory disease rhinoviruses and respiratory disease viral cell recognition and entry human rhinovirus type infection via heparan sulfate is less effi cient and strictly dependent on low endosomal ph many rhinovirus serotypes share the same cellular receptor isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses the major human rhinovirus receptor is icam- a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses cell recognition and entry by rhino-and enteroviruses cdna cloning reveals that the major group rhinovirus receptor on hela cells is intercellular adhesion molecule structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor interferon-gamma (ifngamma) down-regulates the rhinovirus-induced expression of intercellular adhesion molecule- (icam- ) on human airway epithelial cells human rhinovirus type variants use heparan sulfate proteoglycan for cell attachment entry of a heparan sulphate-binding hrv variant strictly depends on dynamin but not on clathrin, caveolin, and fl otillin two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity sequence analysis of human rhinoviruses in the rna-dependent rna polymerase coding region reveals within-species variation phylogenetic analysis of human rhinovirus capsid protein vp and a protease coding sequences confi rms shared genus-like relationships with human enteroviruses virus taxonomy. seventh report of the international committee for the taxonomy of viruses highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study association between human rhinovirus c and severity of acute asthma in children challenges facing real-time pcr characterization of acute respiratory tract infections non-invasive sample collection for respiratory virus testing by multiplex pcr nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses studies with rhinoviruses in volunteers: production of illness, effect of naturally acquired antibody, and demonstration of a protective effect not associated with serum antibody rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults epidemiology of the common cold patterns of illness in rhinovirus infections of military personnel rhinoviruses and respiratory illnesses in university students rhinovirus infections in an industrial population. iv. infections within families of employees during two fall peaks of respiratory illness epidemiology of infections with rhinovirus types and in a group of university of wisconsin student families the seattle virus watch. v. epidemiologic observation of rhinovirus infections, - in families with young children viruses in families spectrum of clinical illness in hospitalized patients with "common cold" virus infections epidemiology of acute lower respiratory disease in children spectrum of viruses and atypical bacteria in intercontinental air travelers with symptoms of acute respiratory infection incidence of respiratory viruses among travelers with a febrile syndrome returning from tropical and subtropical areas acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden acute respiratory illness in an american community: the tecumseh study etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing a modern miasma hypothesis and back-to-school asthma exacerbations seasonal variation in childhood asthma hospitalisations in finland, - descriptive epidemiology of asthma in trinidad, west indies the september epidemic of asthma hospitalization: school children as disease vectors distribution and seasonality of rhinovirus and other respiratory viruses in a crosssection of asthmatic children in trinidad, west indies respiratory infections in the american tropics viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city weekly monitoring of children with asthma for infections and illness during common cold seasons host and viral factors associated with severity of human rhinovirus-associated infant respiratory tract illness rhinovirus transmission one if by air, two if by hand viruses in communityacquired pneumonia in children aged less than years old: high rate of viral coinfection evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections human metapneumovirus in severe respiratory syncytial virus bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis dual respiratory virus infections human metapneumovirus infection in young children hospitalized with respiratory tract disease increased h n infection rate in children with asthma the association of newly identifi ed respiratory viruses with lower respiratory tract infections in korean children comparison of human metapneumovirus, respiratory syncytial virus and infl uenza a virus lower respiratory tract infections in hospitalized young children prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in spain hmpv infections are frequently accompanied by co-infections etiology of communityacquired pneumonia in hospitalized school-aged children: evidence for high prevalence of viral infections the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis rhinovirus infections of children in hospital; isolation of three possibly new rhinovirus serotypes do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? human bocavirus; multisystem detection raises questions about infection community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens rhinovirus-associated hospitalizations in young children a systematic approach to virus-virus interactions induction of endogenous interferon by use of standard live vaccines for prevention of respiratory viral infections potential use of nonpathogenic enteroviruses for control of human disease increased risk of noninfl uenza respiratory virus infections associated with receipt of inactivated infl uenza vaccine some virus isolations from common colds. ii. virus interference in tissue cultures recovery and characterization of non-cytopathogenic rhinoviruses the formation of interferon during acute respiratory virus infection of volunteers broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses comprehensive detection of causative pathogens using real-time pcr to diagnose pediatric community-acquired pneumonia persistence of rhinovirus and enterovirus rna after acute respiratory illness in children prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness presence of viral and bacterial pathogens in the nasopharynx of otitis-prone children. a prospective study chronic rhinoviral infection in lung transplant recipients persistence of rhinovirus rna after asthma exacerbation in children human rhinoviruses: coming in from the cold rhinoviruses are a major cause of wheezing and hospitalization in children less than years of age detection of viruses in human adenoid tissues by use of multiplex pcr clinical severity and molecular typing of human rhinovirus c strains during a fall outbreak affecting hospitalized patients human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection a model for obtaining predictable natural transmission of rhinoviruses in human volunteers parenthood and host resistance to the common cold frequent detection of respiratory viruses without symptoms: toward defi ning clinically relevant cutoff values time lines of infection and disease in human infl uenza: a review of volunteer challenge studies the host response, not the virus, causes the symptoms of the common cold respiratory illness during winter: a cohort study of urban children from temperate australia predominance of rhinovirus in the nose of symptomatic and asymptomatic infants detection of human rhinovirus c viral genome in blood among children with severe respiratory infections in the philippines pneumonia and pericarditis in a child with hrv-c infection: a case report virological studies of sudden, unexplained infant deaths in glasgow - detection of human rhinovirus c in fecal samples of children with gastroenteritis localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization sites of rhinovirus recovery after point inoculation of the upper airway shedding of infected ciliated epithelial cells in rhinovirus colds aerosol transmission of rhinovirus colds transmission of experimental rhinovirus colds in volunteer married couples correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients association between interleukin- concentration in nasal secretions and severity of experimental rhinovirus colds an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study an investigation of the possible transmission of rhinovirus colds through indirect contact mechanisms of transmission of rhinovirus infections role of infectious secretions in the transmission of rhinovirus a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection the complex epidemiology of respiratory virus infections studies of experimental rhinovirus type infections in polar isolation and in england hand-to-hand transmission of rhinovirus colds exhalation of respiratory viruses by breathing, coughing, and talking a new method for sampling and detection of exhaled respiratory virus aerosols detection of airborne rhinovirus and its relation to outdoor air supply in offi ce environments detection of respiratory viruses on air fi lters from aircraft the cold case: are rhinoviruses perfectly adapted pathogens? ifn-g-induced protein is a novel biomarker of rhinovirus-induced asthma exacerbations the type i interferon response during viral infections: a "swot" analysis antiviral innate immunity pathways type iii interferon (ifn) induces a type i ifn-like response in a restricted subset of cells through signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures viral stress-inducible genes rhinovirus induces airway epithelial gene expression through double-stranded rna and ifndependent pathways the interferon response circuit: induction and suppression by pathogenic viruses il- and ifn-alpha regulate the expression of mxa, ′, ′-oas and pkr genes in association with the activation of raf-mek-erk and pi k-akt signal pathways in hepg . . cells the interferon-inducible rna helicase, mda- , is involved in measles virus-induced expression of antiviral cytokines rig-i is cleaved during picornavirus infection human rhinovirus attenuates the type i interferon response by disrupting activation of interferon regulatory factor attenuation of the type i interferon response in cells infected with human rhinovirus mda- is cleaved in poliovirus-infected cells interferons: signaling, antiviral and viral evasion sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion viral infections and atopy in asthma pathogenesis: new rationales for asthma prevention and treatment innate and adaptive immune responses to viral infection and vaccination principles of intracellular viral recognition viruses and toll-like receptors human rhinovirus recognition in non-immune cells is mediated by toll-like receptors and mda- , which trigger a synergetic pro-infl ammatory immune response toll-like receptor function is reduced in adolescents with asthma modulation of the immune system by human rhinoviruses diversity in the bronchial epithelial cell response to infection with different rhinovirus strains host defense function of the airway epithelium in health and disease: clinical background the common cold at the turn of the millennium relationship of viral infections to wheezing illnesses and asthma innate and adaptive immune responses in asthma microrna- limits in vivo immune response-mediated activation of the il- /ifn-gamma pathway, th polarization, and the severity of delayed-type hypersensitivity a defective type response to rhinovirus in atopic asthma acute severe asthma innate immunity in the pathogenesis of virusinduced asthma exacerbations hay fever, hygiene, and household size family size, infection and atopy: the fi rst decade of the "hygiene hypothesis microbial manipulation of immune function for asthma prevention rhinovirus c protease precursors cd and cd' localize to the nuclei of infected cells human rhinovirus a proteinase cleavage sites in eukaryotic initiation factors (eif) gi and eif gii are different rhinovirus a proteinase mediated stimulation of rhinovirus rna translation is additive to the stimulation effected by cellular rna binding proteins human major group rhinoviruses downmodulate the accessory function of monocytes by inducing il- rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production interleukin- : stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness nasal-secretion leukocyte populations determined by fl ow cytometry during acute rhinovirus infection expression of intercellular adhesion molecule- (icam- ) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection? rhinovirus infection of human embryonic lung fi broblasts induces the production of a chemoattractant for polymorphonuclear leukocytes immune responses to viral infections: relevance for asthma the role of nasal secretion and serum antibody in the rhinovirus common cold an elisa for the detection of rhinovirus specifi c antibody in serum and nasal secretion analysis of nasal secretions during experimental rhinovirus upper respiratory infections further characterization of the local respiratory tract antibody response induced by intranasal instillation of inactivated rhinovirus vaccine the time course of the humoral immune response to rhinovirus infection evaluation of an enzyme-linked immunosorbent assay that measures rhinovirusspecifi c antibodies in human sera and nasal secretions effect of rhinovirus infection on cellular immune parameters in allergic and nonallergic subjects rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes antibody to rhinovirus in human sera. ii. heterotypic responses homologous and heterologous resistance to rhinovirus common cold antigenic groupings of rhinovirus serotypes antigenic variation among strains of rhinovirus type hyper-antigenic variation occurs with human rhinovirus type antigenic variation of rhinovirus type isolation of rhinovirus intertypes related to either rhinoviruses and or and is a rhinovirus vaccine possible? allergens, viruses, and asthma exacerbations viruses and bacteria in the etiology of the common cold effect of the infl uenza a/h n pandemic on viral respiratory infections in the fi rst year of life lower respiratory tract infection induced by a genetically modifi ed picornavirus in its natural murine host viruses as precipitants of asthma symptoms ii. physiology and mechanisms infl uenza vaccination: policy versus evidence: no gap between policy and evidence mechanisms of symptoms of the common cold and infl uenza mechanisms of the symptoms of rhinosinusitis kinins are generated in nasal secretions during natural rhinovirus colds european position paper on rhinosinusitis and nasal polyps european position paper on rhinosinusitis and nasal polyps . a summary for otorhinolaryngologists development of a predictive index for picornavirus infections frequent detection of respiratory viruses by real-time pcr in adenoid samples from asymptomatic children rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response prevalence of asthma symptoms and atopic disorders in preschool children and the trend over a decade acute exacerbations of asthma: epidemiology, biology and the exacerbation-prone phenotype gene-environment interactions in asthma viral infections and asthma inception rhinovirus infections more than a common cold community study of role of viral infections in exacerbations of asthma in - year old children respiratory viruses and exacerbations of asthma in adults rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care virus-induced eosinophil mediator release requires antigen-presenting and cd + t cells the etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice role of viral infection and host factors in acute episodes of asthma and chronic bronchitis the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children how viral infections cause exacerbation of airway diseases viruses as precipitants of asthma symptoms. i. epidemiology the childhood origins of asthma (coast) study viral infection in wheezy bronchitis and asthma in children viral specimen collection by parents increases response rate in populationbased virus studies rhinovirus illnesses during infancy predict subsequent childhood wheezing tlr -and th cytokinedependent production of thymic stromal lymphopoietin in human airway epithelial cells role of viruses and bacteria in acute wheezy bronchitis in childhood: a study of sputum greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings asthma exacerbations in children are associated with rhinovirus but not human metapneumovirus infection role of viral infections, atopy and antiviral immunity in the etiology of wheezing exacerbations among children and young adults synergism between allergens and viruses and risk of hospital admission with asthma: case-control study viruses as precipitants of asthmatic attacks in children viral respiratory infections in asthmatic children staying in a mountain resort defective epithelial barrier function in asthma rhinovirus infection-induced alteration of tight junction and adherens junction components in human nasal epithelial cells enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract prevalence and clinical characterization of a newly identifi ed human rhinovirus c species in children with acute respiratory tract infection effect of upper respiratory tract infection on eustachian tube ventilatory function in the preschool child the antiviral compound enviroxime targets the a coding region of rhinovirus and poliovirus importance of respiratory viruses in acute otitis media prevalence of various respiratory viruses in the middle ear during acute otitis media respiratory virus infection as a cause of prolonged symptoms in acute otitis media is acute otitis media a treatable disease? antimicrobials for acute otitis media? a review from the international primary care network a placebo-controlled trial of antimicrobial treatment for acute otitis media a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion role of viruses in middle-ear disease infl uenza vaccination in the prevention of acute otitis media in children effi cacy of a pneumococcal conjugate vaccine against acute otitis media rhinovirus in acute otitis media temporal relationships between colds, upper respiratory viruses detected by polymerase chain reaction, and otitis media in young children followed through a typical cold season presence of viral nucleic acids in the middle ear: acute otitis media pathogen or bystander? rhinovirus in adenoid tissue effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells diagnosis of pathogens in exacerbations of chronic obstructive pulmonary disease overview of virus-induced airway disease virological studies in chronic bronchitis identifying viral infections in vaccinated chronic obstructive pulmonary disease (copd) patients using clinical features and infl ammatory markers. infl uenza other respi viruses viral pneumonia viral pneumoniae in children: incidence and aetiology community-acquired viral pneumonia childhood community-acquired pneumonia community-acquired pneumonia in children etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction diagnosis and management of pneumonia in children viruses and bacteria in sputum samples of children with community-acquired pneumonia a study of pneumonia in a rural area in southern alabama respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner etiology of communityacquired pneumonia in hospitalized children atypical bacterial infections explained by a concomitant virus infection metagenomic analysis of respiratory tract dna viral communities in cystic fi brosis and non-cystic fi brosis individuals evolution of the df cftr mutation the viral aetiology of croup and recurrent croup croup: an -year study in a pediatric practice rhinovirus neutralizing antibody responses and their measurement rhinovirus infection in acute exacerbations of chronic bronchitis: a controlled prospective study respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants rhinovirus bronchiolitis and recurrent wheezing: -year follow-up prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis human rhinoviruses in severe respiratory disease in very low birth weight infants intranasal fl unisolide spray as an adjunct to oral antibiotic therapy for sinusitis respiratory illness caused by picornavirus infection: a review of clinical outcomes rhinitis, sinusitis, and asthma rhinovirus rna in the maxillary sinus epithelium of adult patients with acute sinusitis computed tomographic study of the common cold physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds viral infections of the respiratory tract in patients with cystic fi brosis respiratory infections in cystic fi brosis patients caused by virus, chlamydia and mycoplasma -possible synergism with pseudomonas aeruginosa clinical manifestations of exacerbations of cystic fi brosis associated with nonbacterial infections association of respiratory viral infections with pulmonary deterioration in patients with cystic fi brosis the role of respiratory viruses in cystic fi brosis rhinovirus c and respiratory exacerbations in children with cystic fi brosis infective respiratory exacerbations in young adults with cystic fi brosis: role of viruses and atypical microorganisms a randomized trial of the effi cacy of hand disinfection for prevention of rhinovirus infection effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands effi cacy of organic acids in hand cleansers for prevention of rhinovirus infections virucidal hand treatments for prevention of rhinovirus infection over-the-counter cold medications: a critical review of clinical trials between and ipratropium nasal spray: a new treatment for rhinorrhea in the common cold a -versus -day course of oral corticosteroids for children with asthma exacerbations who are not hospitalised: a randomised controlled trial combined antiviralantimediator treatment for the common cold in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus c protease treatment of picornavirus infections the common cold: control? uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold intranasal interferon-a treatment of experimental rhinoviral colds human tolerance and histopathologic effects of long-term administration of intranasal interferon-a safety and effi cacy of intranasal pirodavir (r ) in experimental rhinovirus infection combating enterovirus replication: state-of-the-art on antiviral research rhinovirus chemotherapy a comparison of the anti-rhinoviral drug binding pocket in hrv and hrv a a new oral rhinovirus inhibitor bta human rhinovirus c protease as a potential target for the development of antiviral agents in vitro resistance studies of rupintrivir, a novel inhibitor of human rhinovirus c protease effi cacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofl oxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells pellino- selectively regulates epithelial cell responses to rhinovirus azithromycin induces antiviral responses in bronchial epithelial cells suggested reading newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord- - f di eu authors: botti, chiara; micillo, alberto; ricci, giuseppe; russo, adolfo; denisco, alberto; cantile, monica; scognamiglio, giosuè; de rosa, antonio; botti, gerardo title: characterization of respiratory infection viruses in hospitalized children from naples province in southern italy date: - - journal: exp ther med doi: . /etm. . sha: doc_id: cord_uid: f di eu most acute respiratory infections (aris) in children are due to viral etiology, and represent an important cause of mortality and morbidity in children < years old in developing countries. the pathogens that cause aris vary geographically and by season, and viruses serve a major role. in the present study, the distribution of the seven respiratory viruses that are more prevalent in southern european countries were retrospectively analyzed in a southern italy hospital, that centralizes pediatric diseases from the naples province. viruses were categorized by a filmarray respiratory panel, and demonstrated no substantial differences in sex, age and seasonal viruses distribution. however, all the investigated viruses had a higher detection rate in the surrounding municipalities than in the metropolitan area of naples. in recent years, the association between air pollution and respiratory infections has become an increasing public health concern. the data in this study support this association in the surrounding areas of naples extensively contaminated by environmental toxic agents. in these areas, characterization of the epidemiology of aris is required to implement a prevention and control program. acute respiratory infections (aris) represent a health issue of great importance, leading cause of mortality in children worldwide, particularly in developing countries ( ) . these represent about % of all diseases in children aged < years ( ) . the major viral agents of ari include influenza a, b, and c viruses (flu), respiratory syncytial virus (rsv), parainfluenza virus (piv), adenovirus (adv), human metapneu-movirus (hmpv), human coronavirus (hcov), and rhinovirus (hrv). the etiology of respiratory diseases is multifactorial and includes, among others, interactions between genetic predisposition and environmental factors ( ) . numerous studies have confirmed that short-and long-term exposures to ambient air pollutants can be associated with a wide range of pathologies, in particular respiratory diseases and cancer. although air pollution has not been shown as the sole cause of respiratory infections, it has been reported that several air pollutants were correlated with increased morbidity of respiratory infections ( ) . in urban areas, irrespective of seasonal frequencies, this correlation increases due to the high incidence of polluting factors. several epidemiological studies have documented a positive association between exposure to particulate air pollution and respiratory symptoms especially among children ( ) ( ) ( ) . it is profusely reported that during the past three decades, large areas of naples county in the southern italy campania region have been extensively contaminated by environmental toxic agents, in particular for the presence of many landfills of industrial wastes. this region was already defined as one of the geographical areas most at risk of neoplastic and respiratory diseases for environmental factors in the report of who (world health organization) in ( ) . although, the epidemiological studies for the association between air pollution and the incidence of respiratory infection in this geographical area are relatively few, virus types identification and their molecular characterization is fundamental not only for surveillance, for diagnostic and therapeutic purposes, but also for highlight the relationships between respiratory diseases in children and concentrations of environment pollutions ( ) . in the present study we characterized the viral spectrum and pattern of aris in children from children's hospital 'santobono pausillipon' which centralizes pediatric diseases of the entire naples province. the aim of this study was to determine the association between respiratory viruses types, patients features (sex, age, season of disease occurrence) and, in particular, geographical origin. patients selection. we retrospectively reviewed the electronic medical records of patients between days and years, with suspected respiratory infection, evaluated at the santobono hospital of naples, between january and january , with filmarray ® respiratory panel (farp) testing on nasopharyngeal swab (nps). from patient electronic medical records the following information was obtained: demographics (age and sex), month of hospitalization, and geographical origin. we have divided the patients into three age groups (early childhood, - years; preschool age, - years; third childhood: - years), in the season of hospitalization (winter: december, january, february; spring: march, april, may; summer: june, july, august; autumn: september, october, november) and geographical origin (metropolitan area of naples and extra-urban areas). filmarray testing. npss were collected according to a standard procedure, kept in viral transport medium, and stored at - ˚c prior to analysis. farp (filmarray ® respiratory panel biofire diagnostics llc ; wakara way salt lake city, ut, usa) is a test based on multiplex pcr. the filmarray rp cartridge is designed for the simultaneous detection and identification of following viruses and bacteria of the upper respiratory tract: influenza a virus (h n , h n , and h n ), influenza b virus, rsv, pivs - , adv, hrv/enterovirus (the assay does not distinguish between these two pathogens), hmpv, hcov ( e, hku , oc , and nl ), mycoplasma pneumoniae, chlamydophila pneumoniae, and bordetella pertussis. the filmarray instrument and pouch system have been described in detail elsewhere ( ) . the research use only version of the filmarray rp system reported a cycle threshold for each positive pcr assay ( ) . statistical analysis. statistical analysis was performed using spss . (spss inc., chicago, il, usa). viral prevalence were compared using the chi-square test for categorical variables, and the cartogram was drawn using excel software (microsoft corporation, redmond, wa, usa). p< . was considered to indicate a statistically significant difference. demographic characteristics. a total of nasopharyngeal swabs were collected and analyzed. in detail, patients were female ( . %) and were male ( . %). most of the patients ( ) were < years old while patients were > years old. patients are heterogeneously distributed in different seasons, while regarding geographical origin, ( . %) patients are of naples metropolitan area, ( . %) originated from neighboring municipalities, and for patients this information is lacking. in our study we considered only viral infections, but we detected also other etiologic agents (bordetella pertussis, chla myd ophila pneu m oniae, m ycopla s m a pneumoniae), highlighted the presence of these infections in patients ( . %). respiratory infection viruses distribution. the total rate of detection of all seven viruses was % ( / ) of patients. in detail, hrv viruses were detected in % of patients, followed by adv viruses ( %), rsv viruses ( %), and piv ( %). a lower incidence has been instead highlighted for flu ( %), hcov ( %) and hmpv ( %) (fig. ) . in a significant proportion of individuals co-infections were also highlighted. in detail, double co-infections were detected in / ( . %) patients, triple in / ( . %) and quadruple infections only in patient (table i) . the more frequent association was detected between hrv and piv viruses / ( %), followed by hrv and adv viruses / ( . %), and hrv and rsv viruses / ( . %) ( table i) . sex and age distribution. viruses appear heterogeneously distributed between sex, with the exception of piv. in detail, adv was slightly higher in males ( / , . %), while flu ( / , . %), hmpv ( / , %) and hrv ( / , . %) were strongly higher in males children (see detection rate fig. ). on the contrary hcov ( / , . %) and rsv ( / , %) are better represented in female patients (see fig. ). regarding age distribution a decline in the incidence of viral infections with age was observed for respiratory viruses, except for flu (fig. ) . in fact the detection rate for flu viruses was lowest in - years patients. the detection rate for adv gradually decrease from - years patients to > years patients. the same trend was highlighted for hcov, piv, hrv and rsv (with a consistently increasing of detection rate in - year-old patients). hmpv was detected prevalently in - year-old children (fig. ) . seasonal distribution. the total detection rate for all respiratory viruses in spring, summer, autumn and winter was calculated. the rates of detection was more heterogeneous during the different seasons. in detail, adv infection was prevalent in summer ( / , . %) and spring ( / , . %) seasons, flu was prevalent in spring ( / , %) and winter ( / , . %), hmpv in winter ( / , %), hcov in autumn/winter ( / , . %), hrv in autumn ( / , . %) and summer ( / , . %), piv in summer ( / , . %) and rsv in winter ( / , %) (fig. ) . geographical distribution. the pediatric patients were divided into two main groups according to origin from the metropolitan of naples and neighboring municipalities. the detection rates for the two areas appear different with a prevalent distribution in extra-urban areas. this appears more evident especially for adv, hrv and rsv viral infections (fig. ) . who reported that ari can represent among the leading causes of mortality in children under years of age. many studies in the literature have described in detail the viruses mainly associated with ari and also their distribution, but showing much conflicting data on populations from different countries ( ) . these variations may be due to socio-economic factors, geographical and climatic differences and efficiency of local health care systems. in recent years, advances in pcr techniques have aided in the rapid and accurate detection of common respiratory pathogens from patient specimens. multiplex pcr can identify and differentiate a large panel of viral and bacterial targets simultaneously and are more rapid and more sensitive methods than cultures or antigen detection ( , ) . in the present report we analyzed the distribution of seven respiratory viruses in a case series of hospitalized patients in metropolitan area of naples and in the neighboring municipalities. the incidence of all analyzed virus is prevalent in early childhood. the distribution of viruses, considered individually, appears to be very heterogeneous, with the largest percentage of hrv in line with other studies on different populations ( , ) . hrv (including rhinovirus and enterovirus) are rna viruses related to picornavirus family ( ) . hrv is associated with the common cold, but can also be implicated in exacerbating asthma attacks and severe complications ( ) . the enteroviruses are categorized into four species, which include a total of serotypes associated with different clinical manifestations ( , ) . in our case series hrv appears more expressed in the male population and its seasonality is mainly associated with summer and autumn, in line with the data present in the literature ( , ) . rsv and adv are, after hrv, the most common virus in our case series, with rsv prevalent among the female children and adv among the male population. rsv is a member of the paramyxoviridae rna viruses family related to human metapneumovirus and pivs. rsv is the most common cause of severe respiratory illness in infants with acute bronchiolitis as a leading cause of hospitalization ( ) . in our case series the winter seasonality of rsv viruses corresponds to the other reported data ( ) . advs are dna viruses uncoated consists of seven species (a-g) and classified by hemagglutination with about serotypes. the species of adv b, c and e cause acute respiratory disease with the main risk factor the long stay indoors ( ) while the adv (species a, d, f and g) can cause cystitis, gastroenteritis and conjunctivitis ( ) . in our study the seasonal distribution of adv is prevalent in summer in line with the literature ( ) . while there are no substantial differences in piv distribution, flu seems prevalent in the male population and their seasonality is consistent with data reported in the literature ( ) . finally, hcov and hmpv viruses are less frequent in our population, with the first more present in male pediatric population and hmpv in female children. coronavirus hcov are characterized serological variants ( e, hku , nl and oc ), and are most commonly associated with infections of the upper respiratory tract ( ) . hmpv belong to the paramyxoviridae family and the infection in newborns and young children is commonly associated with bronchiolitis ( ). it is also widely documented the occurrence of co-infections in some cases. in our case series the more frequent co-infection are between hrv and piv viruses and hrv and adv viruses. these data, in some cases, contrast with other reports ( ) . however, this might be closely related to the geographical location, climate and different social and socio-cultural conditions. co-infections were more common in pediatric patients than in adults as documented by other studies ( ) ( ) ( ) . however, multiple viral infections can be linked to hospital stay, abuse of antibiotic and social conditions, but there are not proves that co-infections can worsen the disease course. our hospital centralizes the majority of patients coming from the metropolitan area of naples but also from neighboring municipalities. our data are interesting for the geographic distribution of patients. in fact all the investigated viruses have a detection rate higher in surrounding municipalities than in the metropolitan area of naples. this is in contrast with most of the data present in the literature, where the prevalence is just in urban areas due to the high presence of pollutants ( ) ( ) ( ) . however, during the past three decades, surrounding areas of naples have been extensively contaminated by environmental toxic agents, in particular for the presence of many landfills of industrial wastes. the most contaminated areas were defined as 'land of fire' ( ) . the relationship between air pollution and respiratory infections has become an increased public health concern in recent years ( ) ( ) ( ) . in fact, the etiology of respiratory diseases is multifactorial and includes, among others, interactions between genetic predisposition and environmental factors as climate change, chemical air pollution and airborne pollens. the short-term respiratory effects of air pollution include decreases in pulmonary function ( ) , increases in inflammatory biomarkers and respiratory symptoms ( ) , infections ( ) , and respiratory mortality ( ) . the environmental risk factors may have an impact on children's respiratory health, above all in urban areas, especially because children inhale more pollutants per kilogram of body weight than adults ( ) . in conclusion, the risk factors between populations can be extremely different, suggesting the need to adequately characterize epidemiology of aris to implement prevention and control program. acute respiratory infections in children aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis chronic obstructive pulmonary disease pulmonary health effects of air pollution locally generated particulate pollution and respiratory symptoms in young children using air pollution based community clusters to explore air pollution health effects in children acute respiratory infections among under-five age group children at urban slums of gulbarga city: a longitudinal study the world health report -conquering suffering, enriching humanity detection and characterization of respiratory viruses causing acute respiratory illness and asthma exacerbation in children during three different seasons filmarray, an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods multiplex pcr: optimization and application in diagnostic virology biofire filmarray respiratory panel for detection of enterovirus d rhinovirus-associated hospitalizations in young children clinical severity of rhinovirus/enterovirus compared to other respiratory viruses in children diagnosis and treatment of rhinovirus respiratory infections epidemiological, molecular, and clinical features of enterovirus respiratory infections in french children between and manual of clinical microbiology epidemiologic, experimental, and clinical links between respiratory syncytial virus infection and asthma abrupt emergence of diverse species b adenoviruses at us military recruit training centers division of viral diseases (dvd) web site prevention and control of seasonal influenza with vaccines; recommendations of the advisory committee on immunization practices--united states clinical disease in children associated with newly described coronavirus subtypes epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china development of three multiplex rt-pcr assays for the detection of respiratory rna viruses viral and atypical bacterial detection in acute respiratory infection in children under five years detection of nine respiratory rna viruses using three multiplex rt-pcr assays incorporating a novel rna internal control transcript air pollution and children's health the role of air pollution in asthma and other pediatric morbidities air pollution, aeroallergens and admissions to pediatric emergency room for respiratory reasons in turin, northwestern italy cancer prevalence in the city of naples: contribution of the gp database analyses to the cancer registries network long-term associations of morbidity with air pollution: a catalogue and synthesis urban air pollution and meteorological factors affect emergency department visits of elderly patients with chronic obstructive pulmonary disease in taiwan the effects of particulate matter on inflammation of respiratory system: differences between male and female air pollution and lung function among susceptible adult subjects: a panel study urban air and tobacco smoke as conditions that increase the risk of oxidative stress and respiratory response in youth fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases revised analyses of the national morbidity, mortality, and air pollution study: mortality among residents of cities oxidative stress in adolescent passive smokers living in urban and rural environments key: cord- -ycc csyh authors: rollinger, judith m.; schmidtke, michaela title: the human rhinovirus: human‐pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery date: - - journal: med res rev doi: . /med. sha: doc_id: cord_uid: ycc csyh as the major etiological agent of the common cold, human rhinoviruses (hrv) cause millions of lost working and school days annually. moreover, clinical studies proved an association between harmless upper respiratory tract infections and more severe diseases e.g. sinusitis, asthma, and chronic obstructive pulmonary disease. both the medicinal and socio‐economic impact of hrv infections and the lack of antiviral drugs substantiate the need for intensive antiviral research. a common structural feature of the approximately hrv serotypes is the icosahedrally shaped capsid formed by identical copies of viral capsid proteins vp ‐ . the capsid protects the single‐stranded, positive sense rna genome of about , bases in length. both structural as well as nonstructural proteins produced during the viral life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, rna and protein synthesis by synthetic or natural compounds. moreover, interferon and phytoceuticals were shown to protect host cells. most of the known inhibitors of hrv replication were discovered as a result of empirical or semi‐empirical screening in cell culture. structure–activity relationship studies are used for hit optimization and lead structure discovery. the increasing structural insight and molecular understanding of viral proteins on the one hand and the advent of innovative computer‐assisted technologies on the other hand have facilitated a rationalized access for the discovery of small chemical entities with antirhinoviral (anti‐hrv) activity. this review will (i) summarize existing structural knowledge about hrv, (ii) focus on mechanisms of anti‐hrv agents from synthetic and natural origin, and (iii) demonstrate strategies for efficient lead structure discovery. © wiley periodicals, inc. med res rev, , no. , – , human rhinoviruses (hrv) are the major cause of upper respiratory tract symptoms, the socalled common colds in humans. their name reflects the primary site of infection. because hrv are nonenveloped, icosahedral viruses of small size with a diameter of about nm ( pico small in latin) that consist of an rna genome, they were assigned to the family picornaviridae. currently, this virus family of the order picornavirales comprises the eight genera enterovirus, hepatovirus, cardiovirus, kobuvirus, teschovirus, erbovirus, aphthovirus, and parechovirus with species and a multitude of serotypes. because of high similarity in genome sequence and genome organization (fig. ) , the former genera rhinovirus and enterovirus have been combined recently, keeping the existing name enterovirus (www.picornastudygroup. com/taxa/species/species.htm). an overview on the current taxonomy of picornaviruses pathogenic for humans as well as on newly proposed species of hrv is given in table i . at present the genus enterovirus includes four approved human enterovirus (hev) species (hev-a, -b, -c, and -d) and two approved hrv species (hrv-a and -b) (www. picornastudygroup.com/taxa/species/-species.htm). since , the global distribution of highly divergent hrv strains was reported. [ ] [ ] [ ] [ ] based on the results of sequence, genomic, and phylogenetic analyses, it was proposed that these strains represent a new hrv species, hrv-c. [ ] [ ] [ ] in , a further proposal concerning a new potential hrv-d species was published after sequencing and analysis of all known hrv genomes. the approved and newly proposed species of the genus enterovirus share z % homology (average amino acid identity) in the precapsid protein p as well as in c and cd. , [ ] [ ] [ ] [ ] different antigenic properties provide the basis for a further division of species into serotypes (table i) . about rhinovirus serotypes are currently known. according to the currently approved taxonomy, most of them ( and hrv hanks) belong to hrv-a and of them to hrv-b. , the genome of all known hrv-a and -b serotypes as well as of several field isolates of hrv-a, -b, and -c has been sequenced completely. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] viruses classified as hrv-c could not be grown in cell culture until now. phylogenetic analyses have been performed with partial sequences, , as well as with the whole genome. , the most recent and comprehensive analysis of all known hrv genomes revealed that (i) hrv-a and hrv-c share a common ancestor, which is a sister group to hrv-b, (ii) hrv-c represents a third species, and (iii) a basal divergence within hrv-a of three distinct strains that led to the proposal of a fourth species hrv-d. hrv-a and -b most often induce a mild, usually self-limited upper respiratory illness in humans characterized by nasal stuffiness and discharge, sneezing, sore throat, and cough. the conventional term is common cold. the common cold is a heterogeneous group of diseases caused by numerous viruses that belong to several different families e.g. rhinoviruses, coronaviruses, enteroviruses, and adenoviruses. but, hrv represent the most common etiological agent worldwide. a large number of distinct strains circulate each year. moreover, in a family or even in a single specimen, multiple hrv serotypes were detected simultaneously. [ ] [ ] [ ] by using rt-pcr and culture, it was shown that hrv induce - % of upper respiratory tract infections in adults as well as children. [ ] [ ] [ ] [ ] [ ] higher incidence has been described from september to november, [ ] [ ] [ ] and from april to may. , in some years and perhaps some geographical areas, spring was a more important time for rhinovirus transmission. , although overall rates of respiratory illness are lower in summer, rhinoviruses are the most frequently isolated at this time of year. the incidence is inversely proportional to age. , by age years, % of the children have antibodies against rhinoviruses. in addition to common cold, hrv are also involved in acute otitis media in children. , moreover, data supporting a causative association with more severe lower respiratory tract infections of infants, elderly persons, and immunocompromised patients have been accumulated. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] studies of childhood and adult asthma have shown that hrv infections can also trigger exacerbations in patients with asthma, [ ] [ ] [ ] [ ] chronic obstructive pulmonary disease, [ ] [ ] [ ] [ ] [ ] [ ] and cystic fibrosis. [ ] [ ] [ ] the recently discovered novel rhinovirus genotype hrv-c was associated with community outbreaks of influenza-like, acute upper respiratory infections and severe low respiratory tract infections of infants e.g. febrile wheeze, bronchiolytis, and asthma exacerbations, which peaked in fall and winter. [ ] [ ] [ ] [ ] [ ] , , in addition, the presence of hrv-c in the middle ear in patients with acute otitis media was demonstrated. hrv spread occurs by means of virus-contaminated respiratory secretions that contain a high virus concentration. [ ] [ ] [ ] besides direct hand-to-hand transmission, small-and largeparticle aerosol transmission of rhinoviruses has been shown. , , children are important ''vectors'' for hrv transmission to family members. moreover, studies with natural hrv-infected adults provided evidence that daily activities of infected people can lead to contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets, and virus transfer to fingers of healthy individuals for infection. , because viral contamination of the hands plays an important role in transmission of hrv from person-to-person, interruption of this step of virus transmission presents a potential target for intervention. this was experimentally proved by treatment of hands by iodine , or salicylic and pyroglutamic acid. observations from experimentally induced infections in normal adult volunteers helped to understand the pathogenesis of hrv infections. [ ] [ ] [ ] [ ] [ ] [ ] the % human infectious dose of rhinovirus is low and the infection rate between and %. after the deposition of hrv on nasal or conjunctival mucosa, viruses are transported to the posterior nasopharynx by mucociliary action of epithelial cells. specific receptors on epithelial cells in the adenoid area are used for binding and entry. already - hr after intranasal inoculation, infectious virus can be detected. virus shedding peaks on the second day after infection and decreases rapidly thereafter. but, small amounts of viruses were discovered in nasal secretions for up to weeks after infection. virus and/or viral rna were demonstrated in the upper as well as lower respiratory tract. , , , using in situ hybridization, arruda et al. ( ) detected viral rna in a low number of ciliated cells in nasal biopsies. in the nasopharynx, a small portion of virus-positive ciliated as well as nonciliated cells was positive for viral rna. in papadopoulos et al. provided evidence that hrv may also lytically infect human bronchial epithelial cells in cell culture as well as in experimentally infected volunteers and induce the production of interleukin- , - , and - . in agreement with these results, hrv rna was detected in - % of children and - % of adults with pneumonia. [ ] [ ] [ ] [ ] taken together, the results of natural cold studies as well as of experimental infection in human volunteers clearly demonstrate that hrv are able to replicate in the upper as well as in the lower airways. hrv infection triggers vasodilation and increased vascular permeability in the nasal mucosa, leading to nasal obstruction and rhinorrhoea. the mechanism is still incompletely understood because no histopathological changes were observed in nasal biopsy specimens from infected persons. this led to the suggestion that clinical symptoms are primarily caused by the inflammatory response of the host to the virus infection and not by the cytopathic effect (cpe) of hrv. results of immunological investigations suggest a modest correlation between the concentrations of il- and il- in nasal secretions and the severity of symptoms in upper and lower hrv-induced respiratory tract disease. , on day - after virus challenge, il- and il- concentrations were significantly greater in nasal secretions from experimentally infected symptomatic subjects than in those from infected asymptomatic or sham-challenged subjects. il- has been proposed as a mediator of neutrophile infiltrations that are observed during symptomatic infections. in experimental rhinovirus infection the onset of symptoms e.g. nasal stuffiness and discharge, sneezing, and cough was observed - hr after intranasal inoculation of the virus. in contrast to rhinovirus infections in adults, fever is found in % of children with upper respiratory tract infections. other symptoms in children and adults may be hoarseness, headache, malaise, and lethargy. sometimes viral infection is accompanied by bacterial complication, leading for instance to acute otitis media in about % of infected children, sinusitis, and pneumonia. [ ] [ ] [ ] , experimental infection was also used to study the causation between rhinovirus infection and asthma as well as copd exacerbations. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] it was shown that hrv infection enhances airway reactivity and predisposes allergic patients to develop late asthmatic reactions. , rhinoviral colds were associated with an increase in histamine responsiveness that was accompanied by a bronchial mucosal lymphocytic and eosinophilic infiltrate. in a recent study, an increased hrv-induced clinical illness severity in asthmatic compared with normal subjects was demonstrated. strong relationships were shown between virus load, lower airway virus-induced inflammation, and asthma exacerbation severity. the results of this study also indicated that augmented th or impaired th or il- immunity are likely important mechanisms. mallia et al. provided evidence that low dose experimental rhinovirus infection in patients with copd induces symptoms and lung function changes typical of an acute exacerbation of copd. viral replication and increased pro-inflammatory cytokine response were associated with symptomatic colds, increases in lower respiratory tract symptoms and reductions in forced expiratory volume in s or peak expiratory flow rate. the epidemiological data and pathology of hrv infections explain their high medical and socio-economic impact. millions of children and adults are taken ill with common cold every year, need medical consultations, are unable to attend school and go to work. , direct costs include hospitalization, medical fees, and symptomatic treatment. moreover, exacerbations are the major cause of asthma and copd morbidity, mortality, and health care costs associated with these diseases. , to date, specific drugs that prevent or reduce rhinovirus infection are not available. common cold can be treated only symptomatically with analgesics, decongestants, antihistamines, or antitussives and antibiotics are often wrongly prescribed. , because of the large number of circulating hrv serotypes, treatment with specific antiviral drugs is considered to be more striking than vaccination. therefore, the search for new highly active synthetic and/or natural anti-hrv compounds is absolutely essential and represents an important area of antiviral research. such an anti-hrv drug would have to be (i) with broad spectrum activity because of the high number of hrv serotypes, (ii) administered very early in infection to demonstrate a good antiviral effect because of the fast infection kinetics, (iii) very safe because of the broad application by millions of people, and (iv) directed against a highly conserved target with low risk of resistance development. due to the very high error rates and the lack of proofreading ability in rna polymerases of picornaviruses, naturally drug-resistant variants may exist in virus populations or resistant viruses can emerge under treatment. as with hiv, another highly variable rna virus, the risk of resistance development and/or selection of resistant virus variants could be minimized by applying combination of drugs directed against different targets. because clinical symptoms are suggested to be primarily caused by the inflammatory response of the host to the virus infection mediated by specific cytokines, a further advantage of drug combinations could be an additional immune-suppressive activity. the knowledge of structural components, nonstructural proteins that are necessary for viral multiplication, and stages of the viral life cycle is an essential precondition for the development of measures to prevent and treat hrv infection. the structure of hrv particles is well known. infectious virions consist of an icosahedral protein shell (capsid) that surrounds and protects the genome, a single positive-stranded rna molecule of approximately , nucleotides. the organization of the enterovirus genome is shown in figure . the viral genomic rna is infectious and encodes a single, long, open reading frame flanked by untranslated regions (utr) at the and end. a small viral protein (vpg) is covalently linked to the end. the end is polyadenylated like cellular messenger rnas. structural components within these utrs e.g. the cloverleaf and the internal ribosome entry site (ires) of the utr play an important role in rna replication as well as protein synthesis. the nucleotide sequence of some regions within these structures is highly conserved among enteroviruses. their blockade could significantly inhibit viral replication. the molecular structure of hrv- a, hrv- , hrv- , hrv- , and hrv- was determined by x-ray crystallography. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the results show that the viral capsid is composed of protomers of each of the three outer structural proteins vp , vp , and vp and of vp in the interior. a star-shaped plateau at the fivefold axis of symmetry, surrounded by a deep depression (canyon) and another smaller depression at the threefold axis were detected. moreover, a hydrophobic pocket was found beneath the canyon floor. with exception of hrv- and hrv- , this pocket is occupied by a fatty acid, the so-called pocket factor. these host cell molecules have been suggested to play an important role in the viral life cycle by providing transient stability to the capsid during its movement from one host cell to another. the outer surface of virions contains neutralization antigenic as well as host cell binding sites. the latter allow the virus to attach to molecules of the host cell membrane (adsorption), the receptors, and to start their life cycle. [ ] [ ] [ ] based on their receptor use, two groups of hrv can be distinguished. the majority of hrv serotypes, the major group uses intercellular adhesion molecule- (icam- ) as their receptor. the viruses belonging to the minor group attach to low density lipoprotein (ldl) receptor, very-ldl (vldl) receptor, and ldlr-related protein on the cells whereat multiple receptors are involved. [ ] [ ] [ ] hrv of the major group apply the canyon as attachment site for binding to icam- . in contrast, ldl receptors of minor group viruses bind near the tip of the five-fold vertex. hrv- has been shown to utilize a sialylated glycoprotein as a cellular receptor. furthermore, a hrv- variant as well as wild-type hrv- can use heparan sulphate proteoglycans for cell attachment in addition to icam- . , the interaction of rhinoviruses with their receptors leads to virus concentration on the cell surface. it induces the release of the pocket factor and conformational changes in the capsid and mediates viral entry via endocytosis. [ ] [ ] [ ] [ ] whereas icam- binding directly causes uncoating, , release of the rna genome from the capsid of ldl-bound minor group rhinoviruses is triggered by acidification of the endosomal, ph-dependent pathway. [ ] [ ] [ ] this detailed knowledge of the capsid structure and function as well as of the virus-receptor interaction offers a good possibility to develop antiviral drugs that interfere with the first steps of the viral life cycle, adsorption as well as uncoating. after uncoating, rhinovirus proteins are synthesized by the translation of a single, open reading frame using cellular ribosomes. the resulting polyprotein of approximately kd is cleaved by viral proteases a pro and c pro into final products ( structural and nonstrutural proteins) immediately after translation. , at first, both proteinases release themselves from the polyprotein by selfcleavage. the primary cleavage of the viral polyprotein between p and p is mediated by a pro . thereafter, cd pro is released from the p precursor by autocatalytic cleavage. next, c pro and its precursor cd pro process proteins of the p (capsid proteins), p , and p (nonstructural proteins) region. interestingly, a pro cleaves also the eif gi/ii component of the translation initiation factor eif f necessary for host cell protein synthesis, [ ] [ ] [ ] and c pro and/or cd pro the rna polymerase transcription factors tfiid, tfiic, sl- , and ubf. therefore, effective inhibition of a pro and c pro would not only inhibit virus replication but could also prevent the shutoff of cellular protein and rna synthesis. moreover, the active site of proteinases is highly conserved among enterovirus serotypes. this high conservation in conjunction with their important role in virus multiplication predestines these enzymes as targets for antiviral therapy. the viral rna polymerase d ( d pol ) represents another very important nonstructural protein of hrv. it forms a complex with both cellular and viral proteins, the rna replication complex. this enzyme synthesizes viral minus-strand rna and uses it as template strand for the synthesis of genomic viral rna. vpg ( b) is the primer for negative-as well positive-strand rna synthesis. negative-strand, but not positive-strand rna synthesis, is stimulated by a pro . further viral accessory proteins include b, c, bc, and ab. besides d pol these proteins can play an important role in inhibition of viral rna synthesis by antiviral compounds. in summary, the knowledge of the structure of the viral capsid, proteases, and polymerase and their important function in the viral life cycle predestine these proteins as potential anti-hrv targets. hrv grow in several human and some primate cells expressing the minor group ldl receptors and/or the major group receptor icam- . human cells susceptible to hrv infections include embryonic kidney, amnion, diploid fibroblasts from embryonic lung, tonsil, liver, intestine, and skin, adult fibroblast lines from aorta and gingival and the kb, hep- , and hela continuous cell lines. but, the susceptibility of hela cells and human fibroblasts to virus infection may vary. hrv multiplication also occurs in primary human airway fibroblasts and differentiated bronchial epithelium. , , the proportion of infectible epithelial cells was shown to be between and %. , but, enhanced levels of viral production were detected in poorly differentiated in comparison to differentiated epithelial cells. the degree of viral infection correlated with il- and il- induction in these cells virus growth causes a typical cpe characterized by ballooning, refractiveness, granularity, and shrinkage of infected cells. the hrv-induced cpe, infectious virus titers, viral protein expression, and rna synthesis can be chosen as parameters to evaluate the anti-hrv activity of compounds in cell-culture based assays. there are several methods for antiviral screening against hrv. the plaque reduction assay has been traditionally performed and accepted as the ''gold standard'' in antiviral testing. [ ] [ ] [ ] however, this test is laborious, time consuming, and the evaluation is subjective. therefore, it is not suited for the routine antiviral testing. it was more and more replaced by methods based on quantification of protection from virus-induced cpe after drug treatment. so, the cpe in sample-treated and untreated cells has been compared by light microscopy. , , but this evaluation is also subjective. another more objective approach is the spectrophotometric quantification of cpe results in neutral red or crystal violet uptake assays, , - and the tetrazolium dye reduction method. , it allows an excellent and rapid antiviral screening of large numbers of compounds using small amounts of extracts, natural, or synthetic compounds. active samples can be scheduled for additional testing using other assays e.g. virus yield or plaque reduction assays, and for studies on the mechanism of action. the activity of potential antiviral drugs has to be approved in vivo. because of the high degree of species-specific variations in icam- preventing infection by major group hrv, practical animal models have been absent for a long time. chimpanzees were infected with several hrv serotypes but without developing clinical signs. hrv do not induce infection in rabbits, guinea pigs, and weanling mice injected with hrv by different routes. one minor group hrv, serotype , was adapted to grow in mouse fibroblasts and used in a mouse model of rhinovirus infection in which growth could be demonstrated. based on the fact that the ldl receptor family is highly conserved between human and mouse, newcomb et al. examined whether hrv- b, another minor group virus, may infect mouse airways in -to -week-old female c bl/ mice. the authors demonstrated that this hrv serotype replicates and induces airway inflammation in vivo. these results strongly correspond to those of bartlett et al. who established three novel mouse models of rhinovirus infection in balb c mice. in the first model, -week-old balb/c mice were infected with hrv- b. in the second model, transgenic balb/c mice, expressing a mouse-human icam- chimera, were inoculated with the major group hrv- . rhinovirus-induced exacerbation of allergic airway inflammation is mimicked in the third model. due to the lack of a small-animal model for hrv infection until , the experimental human challenge model has to be used to approve effects of potential antiviral drugs under controlled conditions in preclinical studies. volunteers were experimentally inoculated with various serotypes e.g. hrv- , hrv- , hrv , hrv- , and hrv- to examine the efficacy of potential antiviral drugs under standardized conditions. [ ] [ ] [ ] [ ] [ ] [ ] examples and results of these studies with capsid-binders, protease, and rna synthesis inhibitors, as well as interferons are described in the following sections. antiviral agents that inhibit virus attachment, capsid uncoating, protein and rna synthesis of picornaviruses are the best studied, , [ ] [ ] [ ] and will be in the focus of this section. inhibition of virus attachment and/or uncoating interrupts the viral life cycle at its beginning and prevents hrv infection. options to prevent these early steps of the viral life cycle include (i) virus neutralization by hrv-specific antibodies, (ii) receptor blockade by antibodies directed against the cellular receptors icam- or ldl, (iii) by soluble receptor molecules, or (iv) by compounds interacting with the viral capsid. because of the high number of serotypes circulating often in parallel, application of hrv-specific antibodies is thought to be no promising approach for prevention or therapy of rhinovirus infection. in contrast, antibodies directed against the cellular receptor or soluble receptor molecules of major or minor group hrv could inhibit and % of hrv serotypes, respectively. therefore, the strategy to prevent virus-receptor interaction by receptor antibodies or soluble receptor molecules has been extensively evaluated in vitro as well as in vivo. the antiviral activity of icam-and ldl-specific antibodies was confirmed in cell culture. , furthermore, the prophylactic effectiveness and safety of intranasally administered rhinovirus murine icam- antibody was assessed in two double-blind, placebo-controlled, randomized studies of volunteers experimentally inoculated with hrv- . in the result, no toxicity related to antibody application was recognized. the higher dosage of mg/subject of rhinovirus murine receptor antibody did not reduce overall infection or illness rates, but was associated with a - day delay in the onset of virus shedding and cold symptoms. viral titers and nasal symptoms were significantly reduced on the second day after challenge. in summary, the monoclonal antibody to the cellular icam- was demonstrated to be not effective enough. a new strategy was the creation of multivalent fab fusion proteins against icam- . a new molecule, named cfy demonstrated a better avidity and in vitro potency against hrv over conventional mabs. cfy is under development as nasal spray with the name of coldsol. antagonism of virus-receptor interaction was considered as another promising way to prevent hrv attachment to host cells. soluble forms of fully or truncated icam- , [ ] [ ] [ ] and ldl or vldl-receptor concatemers [ ] [ ] [ ] exhibited antiviral activity against major and minor group hrv, respectively, in cell culture. soluble forms of icam- compete with receptor binding sites on the virus capsid, hinder an early infection event such as entry or uncoating, or directly inactivate hrv due to the formation of empty capsids. , [ ] [ ] [ ] a soluble ldl receptor fragment neutralized viral infectivity by aggregation. concatemers of the third ligand binding module of the vldl-receptor did not lead to viral aggregation but blocked the receptor binding sites and possibly inhibited viral uncoating by cross-linking the viral capsid subunits via multi-module binding. the antiviral activity of a truncated, soluble form of icam- was proved in hrv- infected chimpanzee. in randomized, double-blind, placebo-controlled trials, the safety and efficacy of intranasal administration of tremacamra, a soluble icam- in experimental hrv- -induced colds in humans, was shown. no further development was reported for these agents. a further option to prevent virus attachment was described for low-molecular-weight compounds, the so-called capsid-binding agents, which enter the small hydrophobic pocket within viral capsid protein beneath the icam-binding canyon of hrv. , zhang et al. showed that drug may integrate into mature viruses by diffusion as well as into progeny viruses during assembly. when hrv- and hrv- were grown in the presence of pleconaril, a higher occupancy occurred than when the drug was introduced into the alreadyassembled viruses. in doing so, capsid-binders induce conformational changes of the canyon of hrv- and hrv- , hinder virus-receptor interactions, and prevent attachment to host cells. , , [ ] [ ] [ ] in addition, uncoating of both hrv serotypes was shown to be inhibited as a result of a potential loss of flexibility of the viral capsid after drug binding. in contrast to hrv- and hrv- , capsid-binding compounds did not prevent attachment of hrv- a. results from x-ray studies showed that drug binding into the hydrophobic pocket of hrv- a replaces the pocket factor but induces only very small conformational changes. therefore, kim et al. suggested that the observed conformational changes are too small to affect receptor binding. but, capsid-binding compounds prevented attachment of hrv- possessing a pocket factor like hrv- a without distinct deformation of the pocket. , , further results from comparative antiviral studies with different capsid-binding compounds and hrv, representative for the major and minor group, did not reveal a correlation between inhibition of adsorption and receptor grouping or antiviral grouping. the reasons for the difference in the mode of action of capsid-binding compounds related to attachment inhibition are not fully understood until now. taken together, inhibition of rna uncoating was found for all investigated serotypes after drug binding independent of receptor grouping whereas prevention of virus attachment was found to be an additional mode of action for individual viruses and/or drugs. till now, various potent compounds belonging to diverse chemical classes have been described as uncoating inhibitors. just to give an impression of diversity, the structures of disoxaril and pleconaril, , - pirodavir and the oxime ether, , , , , the isoxazole derivate compound, , , the imidazole derivative sch , [ ] [ ] [ ] the chalcone ro - , , , dichloroflavan and isoflavan, , the pyridine derivative mdl , , , and the phenoxybenzene mdl- , that exhibit a potent anti-hrv activity (table ii) are shown as examples in figure . they inhibit most of hrv serotypes and a couple of them also affect enteroviruses, however, with varying susceptibility. based on variability of susceptibility to capsid-binders of different length, hrv serotypes were classified into two different groups, a and b. several of the given examples of compounds were also clinically tested. studying the development of clinically effective capsid-binders, the long road to the discovery of a clinically effective anti-hrv drug becomes apparent. one well-described example represents the discovery and optimization of capsid-binders from sterling winthrop pharmaceutical group, the so-called win compounds. first inhibitors originated from juvenile hormone mimetics that demonstrated some activity against hrv- a. determination of the x-ray structure of hrv- helped to understand the compounds' binding sites at the virus capsid. results from subsequent x-ray studies of hrv-win compound complexes revealed the location and nature of binding sites and provided information concerning interactions within these sites. , , this knowledge was used for optimization and design of new compounds. optimized win compounds, for example disoxaril and pleconaril ( fig. ) , consist of a methylisoxazol ring, a substituted phenoxy group, and a five-membered heteroatom ring and inhibit a broad spectrum of rhinoviruses and enteroviruses (table ii) . , [ ] [ ] [ ] in , the first broad-spectrum win compound disoxaril (win ) was tested in clinical trials. the development of crystallurea in human volunteers treated with high doses as well as its low bioavailability ( %) prohibited subsequent development. thereafter, results from sar and qsar analysis were used to further enhance the potency and spectrum of activity. in , another compound, win , was clinically tested. it was not effective in humans infected with hrv- and hrv- . moreover, it was rapidly metabolized and induced a reversible hepatitis. consequently, the further clinical development was stopped. the better understanding of pharmacokinetic properties of capsid binders and synthetic chemistry efforts led to the discovery of pleconaril, an orally bioavailable, welltolerated capsid-binder that inhibits most rhinovirus as well as various enterovirus serotypes. , [ ] [ ] [ ] in , schiff et al. published the efficacy of pleconaril in an experimentally induced coxsackievirus a infection in humans. in phase ii placebo-controlled, natural cold trials, the drug produced a moderate reduction of - . day in the medium time to elevation of illness compared with placebo. these results were confirmed in two subsequent pivotal studies. besides the moderate clinical efficacy, these studies revealed that % of baseline isolates were not susceptible to pleconaril and % developed reduced susceptibility (defined as -fold increase in baseline value). in a subsequent study the relationship of pleconaril susceptibility and clinical outcomes in the treatment of common cold caused by rhinoviruses was demonstrated. based on drug interaction, marginal treatment effect, and possibility of transmission of resistant viruses, the fda did not approve the applied oral administration of pleconaril for the treatment of common cold. the molecular mechanism of drug interaction of orally given pleconaril was shown to be based on hepatic cytochrome p a activation. to reduce adverse effects, shering-plough under license of viropharma completed a phase ii clinical trial with an intranasal formulation of pleconaril for the potential treatment of common cold in high-risk populations in . the results were not published until now. pyridazine analogues developed by janssen research foundation represent another example for the long road to discovery of a clinically effective capsid-binder. in , the broad-spectrum activity of pirodavir ( fig. ; table ii ) against rhinoviruses was published. in the same year the results of a randomized, double-blind, placebo-controlled trail to assess the therapeutic efficacy of intranasal pirodavir in natural common colds were described. possibly as a result of poor water solubility and rapid hydrolysis of pirodavir, no clinical benefit was found. the problem of ester hydrolysis was resolved by the development of oxime ether analogues of pirodavir by biota. an example is shown in figure . like pirodavir these new analogues are potent inhibitors of rhinoviruses. an advantage over pirodavir is their improved bioavailability. bta- , an antiviral analogue with long half-life and good oral bioavailability, was scheduled to a phase ii clinical trial in . the results have not yet been published. in summary, despite extensive research leading to the discovery of potent anti-hrv capsid-binders, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. nearly, the same conclusion has to be drawn for protease inhibitors. because of their pivotal role for viral polyprotein processing and the high conservation of critical amino acids, a pro as well as c pro represent potential anti-hrv targets. results from cell culture-based assays provided evidence that inhibition of hrv replication is in principle possible. for example, processing of the hrv- polyprotein was prevented by pyrrolidine dithiocarbamate treatment in virus-infected hela cells. in contrast to other enteroviruses, [ ] [ ] [ ] [ ] pretreatment of cell monolayers with different nitric oxide donors leading to s-nitrosylation of a pro and c pro had neither an effect on virus replication nor on hrv-induced il- elaboration. the proteolytic activity of a pro of hrv- was specifically inhibited by two elastase-specific inhibitors, and an antiviral peptide representing a derivative of the caspase inhibitor zvad.fmk. , homophthalimides, e.g. ly ( fig. ; table ii) , were described as inhibitors of a pro as well as c pro . in contrast to protease c, no structure-activity relationship studies have been reported for hrv a protease. moreover, protease a accomplishes only one cleavage in hrv polyprotein, while protease c performs all other cleavages. after elucidation of the crystal structure of c pro , computer modeling of structural features of protease inhibitors became possible. furthermore, structure-based design was used to develop mechanism-based inhibitors of the c protease with potent antiviral activity against multiple hrv serotypes. , highly active compounds incorporate various michael acceptor moieties, irreversibly bind to c pro , and exhibit anti-hrv- activity in hela cells. , structure-activity studies were performed to optimize protease inhibitors. , [ ] [ ] [ ] [ ] [ ] these efforts resulted in the identification of a highly active anti-hrv compound, ag (rupintrivir; fig. ; table ii ) that entered clinical trials. in cell culture, ag inhibited a broad spectrum of laboratory hrv as well as clinical isolates. , , in a single-cycle, time-of-addition assay it demonstrated antiviral activity when added up to hr after infection. inhibition of hrv replication strongly correlated with reduction in the level of il- and il- release into cell supernatant, leading to the suggestion that this agent may not only block virus replication but also diminish symptoms. the pharmacokinetics and safety of rupintrivir were proved in two double-blind, randomized, placebo-controlled studies. intranasal rupintrivir, administered as single doses of and mg or every hr, six times per day, for days, was safe and well tolerated. three double blind, placebo-controlled clinical trials were conducted to assess rupintrivir nasal spray ( % solution) for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. rupintrivir prophylaxis reduced the proportion of subjects with positive viral culture by % and viral titers but did not decrease the frequency of colds. drug treatment led to the reduction of the mean total daily symptom score by %. subjects receiving rupintrivir also demonstrated significantly lower viral titers and rna levels than placebo-treated subjects on days , , and and on days and , respectively. there was no influence on the proportion of subjects with positive viral culture and the frequency of colds. clinical development was terminated because rupintrivir did not act in a subsequent natural infection study in patients. in parallel research efforts, an orally bioavailable inhibitor of table ii) , was discovered. , like rupintrivir, this compound is an irreversible inhibitor incorporating a michael acceptor moiety that forms a covalent bond with the c protease active site cysteine. it demonstrated an antiviral activity against all hrv and related picornaviruses tested. in a phase clinical study, compound was shown to be safe and well tolerated. according to a publication of patick, no further clinical development was planned for this compound. the blocking of viral rna synthesis during replication represents another site for chemotherapeutic interdiction. it was shown that, rhinoviral rna can be targeted in a sequence-specific manner by deoxyribozymes, morpholino oligomers, and small interfering ribonucleic acids. the efficacy of the latter two approaches was confirmed in cell culture. in addition, -furylmercury chloride ( fig. ; table ii ), table iii) , and pyrrolidine dithiocarbamate (fig. ) interfered with rhinoviral rna synthesis and inhibited hrv replication in cell culture-based assays. , the nucleoside analog ribavirin ( fig. ; table ii ) that inhibits a broad spectrum of rna as well as dna viruses acts also against hrv- in hela cells. , the cellular inosine monophosphate dehydrogenase that controls de novo synthesis of purine nucleosides represents the principal target in the mode of action of ribavirin. moreover, when ribavirin is incorporated into picornavirus rna, it pairs equally well with either uracil or cytosine inducing mutations that can be lethal to rna viruses. further identified mechanisms of action for ribavirin include inhibition of genomic rna capping, enhancement of host t-cellmediated immunity against viral infections through helping to switch the host t-cell phenotype from type to type . another compound with potent anti-hrv activity in vitro is enviroxime ( fig. ; table ii ), a benzimidazole derivative. , it inhibits viral plus strand rna synthesis. in particular the a protein, which is involved in the initiation of plus strand rna synthesis, was implicated as likely target of drug activity. , however, results from another study suggest that enviroxime targets a complex of proteins and/or cellular factors and that the exact mechanism remains to be studied. although there was a statistically significant reduction in clinical score in a prophylactic study with hrv- -infected volunteers, enviroxime failed in experimentally induced hrv- and hrv- infection, , and in clinical studies, - because of poor bioavailability and side effects. in an attempt to overcome the marked hydrophobicity, water insolubility, and toxicity, wyde et al. incorporated enviroxime into liposomes and then tested the anti-hrv activity and toxicity of the liposome-incorporated enviroxime in cell culture. the liposome preparation of enviroxime inhibited hrv- a and hrv as effective as the parent compound and was -to z -fold less toxic. in contrast to free enviroxime, the liposome preparation was readily and successfully delivered by small-particle aerosol to the upper and lower respiratory tract of mice. in another attempt to overcome the disadvantages of enviroxime, several benzimidazole as well as nonbenzimidazole analogs were synthesized and studied. [ ] [ ] [ ] [ ] even though some compounds were better bioavailable and could be administered orally, none of these compounds was tested in clinical studies. besides virus-specific targets, cellular inhibitors like interferons may represent a therapeutical approach. among other activities, interferons exhibit antiviral activity. the advantages of interferon application include the broad spectrum of activity and low risk of resistance development. human leukocyte and fibroblast as well as recombinant human a interferons prevent the hrv-induced cpe in cell culture whereas a variation in sensitivity was observed. [ ] [ ] [ ] intranasally applied recombinant interferon a and interferon b have been shown to be effective in humans when provided prophylactically both in experimental and natural rhinovirus colds. [ ] [ ] [ ] [ ] [ ] [ ] significant reductions in illness frequency, mean symptom score, nasal secretion weights, and frequency of virus isolation were observed. in contrast, recombinant interferon g did not prevent hrv infection or illness and may enhance the symptoms. little to no therapeutic effect was found in patients with common cold after interferon treatment. , moreover, blood-tinged mucus and nasal bleeding were described as side effects. , combining interferons with dichloroflavan, enviroxime, chalcone ro- - produced synergistic increases in antiviral activity in vitro against hrv- and hrv- . an attempt to demonstrate synergy between the anti-hrv effect of recombinant human rhuifn a and enviroxime in hrv- and hrv- -infected volunteers failed. according to the authors, the main reason for this failure may be the rapid removal of enviroxime from the nose when given intranasally. a. impact of natural products nature provides an astonishing pool of secondary metabolites biosynthesized from living organisms such as plants, fungi, protozoan, insects, and other animal sources. in contrast to synthetic compounds, natural products are characterized by an overwhelming chemical diversity. previously, the chemical diversity space between these two groups was evaluated with respect to drug substances by feher and schmidt. it is shown that combinatorial compounds densely populate a small area, whereas natural products cover a wider range quite similar to the chemical space occupied by drug substances. the authors accordingly suggest that combinatorial libraries that mimic the distribution properties of natural products might be more biologically relevant. one may assume that secondary metabolites evolved as reaction to their target receptors related to defence, protection, attraction, and signalling. these adaptation processes have enriched not only the metabolites' structural diversity but have also optimized drug-like metabolic traits likely to have favorable pharmacokinetic properties. , it is this evolutionary concept that gives the pool of natural products the greatest source of scaffold diversity with molecules of biological relevance. newman and cragg analyzed the number of drugs approved between and and circumstantiated that especially the anti-infective area is strongly dependent on natural products and structures derived from natural scaffolds. the anti-infectives including the antiviral vaccines are with . % or launched drugs by far the major category with only about % being synthetic in origin. from to , vaccines and antiviral drugs have been approved. excluding the high number of vaccines ( ) and biologicals ( ) , most of the small antiviral molecules are based on nucleoside structures or on peptidomimetics; only . % are classified as totally synthetic drugs. however, till now, neither a synthetic nor a naturally derived anti-hrv drug substance has been approved for the treatment or prevention of hrv infections. intensive research and development efforts in the field of natural products revealed several inhibitors of viral attachment and entry, and inhibitors of viral protease from natural sources. the efficacy of natural products is not only reflected by statistics of launched drugs but also by empirical knowledge gained over centuries by successful application of naturalbased ethnomedicinal products such as plants, culinary herbs, and spices. phytochemical and pharmacological work performed with ethnomedicinal anti-hrvs mainly from plants revealed a high number of active metabolites from different chemical classes, e.g. coumarins, flavonoids, alkaloids, quinones, terpenoids, polyphenols, and polysaccharides. natural products include complex extracts and their chemical entities, which are biosynthesized by nature. for an unambiguous presentation of anti-hrv natural products it is of prior importance to first distinguish between a single chemical entity from nature, i.e. an isolated, purified natural compound, on one hand, and a natural preparation comprising hundreds to thousands of constituents, mainly secondary metabolites, on the other hand. if natural preparations are derived from plants, they might also be labelled as botanicals, phytoceuticals, or phytotherapeutic agents. these multicomponent preparations might show a varying profile of their constituents depending on the used species, origin, collection time, plant parts, extraction procedures, preparation methods, and manufacturing processes, just to mention a few important elements. these parameters affect the final product in terms of the qualitative and quantitative composition of chemical constituents, which may have an impact in biological activity. accordingly, studies performed with phytochemically not specified extracts or nonstandardized preparations often suffer from irreproducible and incomparable results a wide variety of natural preparations showed to be acting therapeutically in hrv and other viral infections with often complementary and overlapping antiviral mechanisms of action. [ ] [ ] [ ] most of these remedies are described in ethnopharmacological sources or handed down for generations. they usually consist of simply prepared natural items whose chemical composition is complex. many of the contained secondary metabolites, possibly active principles, have never been examined chemically or biochemically using modern medical knowledge. they are however components of plant medicines, which have stood the test of time and as such may offer clues of great interest to medicinal chemists. a clear advantage of the application of these products is their absent or relatively low toxicity due to a usually long-term empirical trial. although the knowledge of the immuno-pathogenesis of rv-induced diseases remains limited, the host defense function of the airway epithelium plays an important role in the innate-immune response to hrv-infection. host cells respond by the production of mediators with antiviral activity such as type i interferons and nitric oxide, and produce cytokines and chemokines that influence the subsequent induced innate-and specific-immune response. these processes are beneficial in facilitating clearance of virus from the respiratory tract, but also cause immuno-pathology. following hrv-infection, disease severity is dependent on direct, harmful effects of the virus as well as tissue damage as a result of the host antiviral immune response. accordingly, a number of agents with phenomenological effects against common cold have shown to exert their activity more in the field of regenerating tissue damage than on a direct anti-hrv effect. several herbal remedies consisting of a multitude of secondary metabolites from different chemical classes may attribute in a beneficial way for the treatment of common cold by reducing symptom severity and duration due to their immune-modulating, anti-oxidative, and anti-inflammatory properties. beside these commonly observed bioactivities of natural products, multicomponent mixtures like botanicals often show overlapping symptomatic effects as well as synergistic and/or additive properties. thus, it is a challenging endeavor to track down an observed phenomenological effect of a complex mixture on a molecular level. the following section explores the significance and current knowledge of selected botanicals for the prevention and therapy of common cold. questions about ( ) clinical evidence of efficacy, ( ) the constituents or at least the chemical classes that are involved in the observed anti-hrv effect, and ( ) the involved pharmacological targets were covered as far as possible. echinacea preparations include expressed juice from aerial parts as well as extracts of roots or aerial parts, or both, from one or more species of the genus echinacea (e. angustifolia, e. purpurea, and e. pallida). they are the most recognized botanicals for prevention and treatment of common cold and flu, and account for the second top-selling herbal products in the us-market. accordingly, echinacea has come under much scientific scrutiny. the high number of studies dealing with the effectiveness of echinacea for preventing and treating the common cold from clinical trials was recently reviewed by woelkart et al. the authors summarized the findings of the meta-analyses regarding the randomized controlled trials evaluated in the cochrane database, the randomized clinical trials analyzed by shah et al., and the experimental hrv-infection studies pooled by schoop et al. to sum up, the clinical data on echinacea so far are not fully consistent, mainly based on problems inherent in assessing the efficacy of echinacea preparations, such as lack of comparability of available preparations, study design, and outcome. nevertheless, the meta-analyses showed some evidence that preparations based on the aerial parts of echinacea purpurea might be effective for the early treatment of colds in adults. echinacea showed to decrease the odds of developing the common cold by % and the duration of a cold by . days. similarly, the evaluation of three induced rhinovirus prevention studies revealed the odds of experiencing a clinical cold were % higher with placebo than with echinacea. stepping into a molecular level, several constituents found in echinacea species could potentially affect the symptoms of common cold. chemically identified substances include polysaccharides and glycoproteins, caffeic acid derivatives (especially cichoric acid and echinacoside), and lipophilic polyacetylenes and alkamides. pharmacological studies have shown that cichoric acid, alkamides, glycoproteins, and polysaccharides possess immunomodulatory activity. additionally, alkamides have been reported to exert not only antiinflammatory effects but also cannabinomimetic properties, which are suggested as molecular mode of action of echinacea alkamides as immunomodulatory agents. raduner et al. showed that some echinacea alkamides exert cannabinoid type receptor-dependent and independent immunomodulatory effects on cytokine expression. different echinacea constituents were evaluated for their anti-oxidative effects measuring the inhibition of in vitro cu(ii)-catalyzed oxidation of human low-density lipoprotein. thereby, the major caffeic acid derivatives, cichoric acid and echinacoside, showed the highest anti-oxidative effects, which was even higher when combined with a natural mixture of alkamides. sharma et al. used cytokine antibody arrays to investigate the changes in the pro-inflammatory cytokines and chemokines released from human bronchial epithelial cells exposed to hrv . application of two chemically characterized echinacea extracts showed a reversion of the stimulated release of numerous pro-inflammatory cytokine-related molecules, e.g. for the cytokine il- , and the chemokines il- and eotaxin. in a similar study, an echinacea extract rich in polysaccharides and another rich in alkamides and caffeic acid derivatives were as well able to neutralize the effects of hrv-infected epithelial cells. using gene expression analysis both studies revealed the anti-hrv benefit of echinacea preparations being involved in multiple immune response signaling pathways. taken together, the numerous pharmacological findings from literature, the potential of echinacea preparations, and their constituents to combat or prevent common cold can be deduced to immune modulating, anti-inflammatory, and anti-oxidative properties that may also act in some combination of these event, rather than acting directly on hrv. garlic cloves have been used traditionally to treat a number of infectious diseases. however, only few confirmatory studies have been published regarding the traditional antiviral uses. the clinical effectiveness of garlic on the prevention of common cold was investigated by josling in , who published a double-blind, placebo controlled study assessing patients more than a -week treatment period with an allicin-containing garlic supplement. common cold infections and symptoms were recorded in a daily diary. patients in the treatment group had significantly fewer colds than patients in the placebo group ( vs. , po . ) who also had a longer duration of symptoms ( . vs. . days, po . ). as soon as the garlic is chewed, cut, or pressed, its main ingredient, the sulphur containing alliin, is broken down by the enzyme alliinase to the thiosulfinate allicin. by steam distillation allicin is transformed to diallyl disulfide and diallyl trisulfide that are responsible for the distinctive smell of garlic. further, allicin transformation compounds, such as e-and z-ajoene, are not found in fresh garlic, but in lipophilic extracts. by investigation of different garlic extracts and isolates against a number of different human pathological viruses, weber et al. could show that allicin was the most active virucidal component from fresh garlic and fresh extracts. results of the direct pre-hrv- -infection incubation assay let suggest allicin to bind to the viral protein capsid, leading to a subsequent inhibition of viral adsorption and penetration. although the garlic thiosulfinates are endowed with significant cytotoxicity, the antiviral effects were obtained in nontoxic concentrations. beside the direct anti-hrv effect of fresh garlic extract and allicin, a number of human immune functions were found to be enhanced in vitro by aqueous garlic extract, its polar, and thiosulfinate fractions. in north america, panax quinquefolium, the ginseng species indigenous to both canada and the united states, has been a popular herbal remedy to combat stress, and to modulate both natural and acquired immune responses. american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, have been found efficacious in the prevention of upper respiratory infections in immunocompetent healthy adults. , in a randomized, double-blind, placebo controlled trial, mg of a proprietary american ginseng root extract was given to community-dwelling elderly adults (age year) twice a day more than a ''cold and flu'' season period of months. one month into the study, all participants received an influenza vaccination. during the first two months, no significant differences in duration and incidence were observed when compared to placebo. however, during the last two months significantly fewer subjects of the ginseng group reported acute respiratory syndromes than the placebo group ( vs. %). additionally, the duration of respiratory symptoms was reduced by % in the ginseng group. in a similarly arranged trial, healthy adults (ages - years) with a history of at least two colds the previous year commenced a month study at the beginning of a cold and flu season. they received two mg capsules daily of standardized american ginseng root extract or a placebo. outcomes measured were number of colds including symptom severity and total number of symptomatic days. a therapeutic effect was reported regarding symptom severity and fewer symptom days that were and . % lower in the ginseng group than in the placebo group. a phase ii randomized, controlled trial of dosing schedules of american ginseng root extracts, rich in poly-furanosyl-pyranosyl-saccharides, evaluated the safety, tolerability, and efficacy in a pediatric population already suffering from an upper respiratory tract infection. the results showed no serious adverse events and a good tolerability of both ginseng doses; however, frequency and severity of symptoms were not significantly different among each of the three treatment groups, i.e. standard dose, low dose, and placebo. the most prominent constituents of the genus panax are the triterpene saponins ginsenosides. they are known to have numerous pharmacological activities such as anti-cancer, anti-diabetes, antiviral, and anti-atherosclerosis effects. some compounds of this chemical class showed to be responsible for the immunostimulant activity of ginseng. on the other hand, the efficacy of a polysaccharide-rich extract of american ginseng was compared with an extract rich in ginsenosides on systemic and gut-associated immune function. the authors of this study investigated the lymphocytes isolated from spleen, mesenteric lymph nodes and peyer's patches, and immune cell proportions and cytokine production from sprague-dawley rats. they could show that the polysaccharide-rich ginseng extract modifies the rats' systemic immune responses and affects the gut-associated immunity in a manner distinct from that of the ginsenoside-containing extract of american ginseng. a direct antiviral activity of ginseng constituents could be attested for the polysaccharides on rotavirus infection in ma cells. the triterpene saponins, however, did not exhibit any rotavirus infection-inhibitory activity in this study. a moderate in vitro virucidal effect (id mm) of the ginseng saponin chikusetsusaponin iii against herpes simplex virus type i was detected by fukushima et al. this compound exhibited an intracellular inhibitory activity, but could only marginally affect the viral proteins postinfection. the ancient chinese formula bu-zhong-yi-qi-tang (japanese name hochu-ekki-to) is a traditional herbal medicine in china and japan that is composed of ten species of medicinal plants, namely astragali radix, atractylodis lanceae rhizoma, ginseng radix, angelicae radix, bupleuri radix, zizyphi fructus, aurantii nobilis pericarpium, glycyrrhizae radix, cimicifugae rhizoma, and zingiberis rhizoma. this formula is reported to have various immunomodulatory, [ ] [ ] [ ] and anti-inflammatory activities. yamaya et al. recently investigated the effects of hochu-ekki-to in cultures of human airway epithelial cells infected with hrv- . the output of virus, associated levels of viral rna, and the production of icam- , cytokines and acidic endosomes in cells were measured. in airway epithelial cells hochu-ekki-to was able to decrease virus output and susceptibility to hrv infection by decreasing icam- and by blocking the entry of viral rna into the cytoplasm from the endosomes. glycyrrhizin, a major component of one herbal ingredient of hochu-ekki-to, i.e. glycyrrhiza glabra, was able to reduce supernatant virus titers dose-dependently, with a maximum effect between . and . mm. however, no clinical trials with representative numbers of subjects are published so far. the human rhinovirus k . umckaloabo (pelargonium sidoides) p. sidoides and p. reniforme form the origin of the popular drug umckaloabo. this herbal remedy from south africa has found entrance in western medicine mainly as aqueous ethanolic root extract from p. sidoides for the treatment of infections of the respiratory tract. the efficacy of umckaloabo compared with placebo has been evaluated in adults suffering from common cold by lizogub et al. the applied herbal preparation was well tolerated by the patients. the study demonstrated only a weak efficacy of umckaloabo compared to placebo after days. after days, however, the p. sidoides extract significantly reduced the severity of symptoms and shortened the duration of the common cold compared with placebo. just recently, timmer et al. selected randomized controlled trials examining the efficacy of p. sidoides preparations for the treatment of various acute respiratory infections and analyzed their efficacy and safety. the authors concluded that umckaloabo may be effective in alleviating symptoms of acute rhino-sinusitis and the common cold in adults. it may be effective in relieving symptoms in acute bronchitis in adults and children, and sinusitis in adults. reliable data on the treatment for other acute respiratory infections however were not obtained. identification of the metabolites from umckaloabo revealed a high number of different chemical classes, such as phenolic and cinnamic acids, tannins, flavonoids, and coumarins. antibacterial activities of umckaloabo against different pathogens have been reported. phenols, coumarins, and tannins have been identified to contribute with moderate antibacterial activities, however, cannot explain the effect of the whole extract (reviewed by kolodziej , ). additionally, p. sidoides extracts have been reported to significantly activate the nonspecific immune system by induction of tnf and no-release, and ifn-like activities. these effects are assumed to contribute to the controversially discussed potential of p. sidoides extract for the treatment of upper respiratory tract infections. only one study reports a direct antiviral effect, i.e. a clear dose-dependent anti-herpes simplex virus acitivity for the aqueous root extract of p. sidoides. further pharmacological studies are needed to elucidate potential direct anti-hrv properties of umckaloaba and its constituents. carrageenan, a mixture of different polysaccharides, which is mainly extracted from red seaweeds, has been extensively used in food, cosmetic and pharmaceutical industry as a thickener and gelling agent. it has previously shown an antiviral efficacy against several viruses. , in a recently published study, lambda-, kappa, and iota-carrageenan were investigated for their anti-hrv inhibiting potential. at a concentration of mg/ml iotacarragenan, a sulphated polysaccharide, was able to fully inhibit virus-induced cell death in hrv- infected hela cells. based on their studies, grassauer et al. concluded that iotacarrageenan is effective against different hrv-serotypes on primary human epithelial cells. it is hypothesized by the authors that iota-carrageenan might create a hostile environment for hrv and thereby block viral entry and replication. because of its safe application and proved in vitro efficacy, iota-carrageenan deserves consideration as a candidate for clinical trials for prevention and therapy of hrv-induced common cold. the level of knowledge on the impact of the six botanicals on hrv-infection discussed above is different and heterogeneous. the best studied herbal remedy associated with common cold, i.e. echinacea, showcases the innate problem connected with multi-component mixtures: starting from the late nineties till june some original articles have been published to this topic and tried to elucidate questions concerning efficacy, molecular mechanism, and bioactive ingredients of echinacea. although some evidence is provided for the effects of extracts, chemical classes as well as well-defined constituents on specific targets and pathways, the findings cannot be deduced to a common denominator. further, results from clinical trials often suffer from lack of comparability, because of using different study designs, outcome measures, and overall the application of different preparations. a proper quality analysis and characterization of the preparation under investigation is mandatory and should follow the recommendations and guidelines for reporting clinical trials for herbal medicine. as underlined before (sections . . and . .), the chemical complexity of a natural preparation might be beneficial in terms of synergistic, additive, and overlapping effects caused by the multitude of evolutionary trimmed metabolites, which may attribute with modulating multi-target effects. on the other side, exactly this fact is hardly compatible with the proper assignment of an activity to a defined chemical entity according to western medical practise. in contrast to a single compound (synthetic or naturally based), the chemistry of a botanical is not only complex but also varying. the analytical profile and in term the pharmacological profile of the investigated samples can differ substantially. accordingly, the quantitative and qualitative comparison of different studies resulting from botanicals is by far more complex than those performed with pure single compounds, and may also explain why so little emphasis from pharmaceutical industry has been put into the further development of even promising natural preparations. in general, the search for potent, selective, nontoxic compounds that might be developed further to a drug substance is a multidisciplinary, time-and cost-consuming process. therefore, strategies for a target-oriented discovery of lead-structures either from nature or synthesis are in high demand. some of them will be discussed in the following section, providing examples from anti-hrv research. as already mentioned, nature provides an extremely rich pool of bioactive natural products. it is however a challenging endeavor to find exactly those compounds that show an activity on the focused target. in natural product research hints from folk medicine are a valuable starting point to dig for lead structures of certain interest. the majority of active principles from higher plants has been discovered as a result of ethnopharmacologically directed pharmacognostic research. , oral or written indications for a beneficial application of a natural material first need a critical evaluation as to the selection of the correct material, its medicinal preparation, the kind of application, and overall the pharmacological profile. a rational criticism of often anecdotal efficacy from traditional medicine is a mandatory attitude to avoid overinterpretation of handed-down information. in case of pretended anti-hrv remedies, the reported biological efficacy obviously suffers from being restricted to respiratory diseases, which might be caused by a panel of bacterial or viral infections. thus, an approved remedy may affect the microbes, or show an immune modulating or even antiinflammatory effect, or a combination of these. the holistic access is an innate character of ethnopharmacology and needs to be tracked down to the specifically involved target/s. in a recently published study focusing on the discovery of hrv-capsid binders from nature using a pharmacophore-based virtual screening of an ethnopharmacologically biased d-multiconformational database, some secondary metabolites were predicted to act as capsid-binding inhibitors of hrv. for an in depth phytochemical and pharmacological investigation, it was mandatory to focus on one promising natural material. thus, we consulted the ethnobotanical source materia medica, which was written by pedanius dioscorides in the st century ad. the treatise consisting of five books comprises some , or so drugs derived from minerals, animals, and the majority of them from plants ( ). it represents a great repository of botanical, medical, and pharmacological lore. scrutinizing the natural materials underlying the obtained virtual hits we came across asafetida, which is a gum resin gained from the roots of a variety of foul-smelling ferula species from the apiaceae family. based on the descriptions given in the materia medica there is strong evidence that the juice (i.e. resin) of the popular ancient silphion originating from media and syria corresponds to asafetida (book iii, cap. ). yielded by incision of the root and stalk and frequently mixed with sagapenon, i.e. the resin of f. persica willd (book iii, cap. ), it is reported to be effective in the context of upper respiratory diseases, e.g. ''for chronic harshness of the throat,'' ''it clears the voice,'' ''shrinks the uvulas,'' ''suitable for a cough,'' ''for pleurisy,'' ''for chest pain;'' sagapenon is described as follows: ''it clears thick matters from the lungs,'' ''given to those who are chilled.'' these descriptions finally helped to prioritize those virtual hits, which have been reported to be constituents of asafetida. the pharmacological investigation of asafetida and its constituents farnesiferol b and c ( fig. ; table iii ) revealed a distinct anti-hrv- effect in the low micromolar range using a cpe inhibitory assay. the results of this study provided a rationale for the ancient usage of asafetida for upper respiratory tract infections. on the other hand, the traditionally manifested evidence for asafetida for the treatment of common cold symptoms substantially helped in the selection of this plant material in the search for anti-hrv capsid binders. typically, an ethnopharmacologically based discovery of an active (anti-hrv) extract is followed by a bioassay guided fractionation, and the isolation of those constituents that are responsible for the extracts' bioactivity. the concept of a bioassay-guided approach is the fractionation accompanied by simultaneous detection of the activity during the separation steps, which results in a continuous enrichment, and finally in the isolation of the active ingredient/s. in this way a large number of anti-hrv agents from natural sources have already been discovered. semple et al. investigated the active principle of the asteraceae plant pterocaulon sphacelatum, which has been used in traditional medicine of aboriginal people of australia as a favored treatment for respiratory infections, especially colds. by means of an antiviral activity-guided fractionation measuring the poliovirus-induced cpe assay, the authors identified the flavonoid , , -trimethoxy- , , -trihydroxyflavone, i.e. chrysosplenol c ( fig. ; table iii ) as potent and specific inhibitor of the picornaviral replication. , , -trimethoxycoumarin ( fig. ; table iii) was also isolated as a major constituent from the ethanolic extract of p. sphacelatum, but showed no activity against poliovirus. interestingly, this coumarin exhibited a significant effect in the hrv- -induced cpe assay in a recently performed virtual parallel screening study performed in our laboratory. in contrast to the isolated coumarin, chrysosplenol c was already known as a member of the -hydroxy- -methoxyflavones, which represent potent and specific inhibitors of picornaviral, especially rhinoviral replication. [ ] [ ] [ ] in , vanden berghe, haemers, and vlietinck provided a profound survey of antiviral agents from higher plants, and demonstrated the impact of ethnobotanical knowledge in their search for antiviral compounds from african medicinal plants. the selection of investigated plant species was mainly based upon their use in the treatment of viral diseases by african traditional healers. the antiviral activity of different plant species belonging to families was investigated; thereof species exhibited prominent antiviral properties against one or more of the tested viruses. the most pronounced activity against picornaviruses was recorded within the genus euphorbia. all compounds detected as antiviral constituents from the respective extracts were identified as -methoxyflavone derivatives, especially -methylethers of quercetin and kaempferol ( fig. ; table iii ). they showed no significant cytotoxicity and were highly active in tissue culture against all human picornaviruses. in tissue culture, cells infected with different picornaviruses (among them also hrv) no cpe was observed, when cells had been treated with mg/ml of these -methoxyflavones. the selection of natural materials based on ethnopharmacology is a profound rationale for lead structure discovery and highly superior to random selection. this however rarely applies to marine organisms, fungi, and microbes. although these organisms are esteemed as highly valuable source for bioactive metabolites, hardly any records from folk medicine are given for them. a medium-sized activity screening using a robust cell-based or in vitro-assay with reasonable effort as to time and costs is a strategy to get a first insight into the antiviral activities of extracts, fractions, and compounds from synthesis or nature. for the discovery of novel naturally based hrv c-protease inhibitors, singh et al. used a small peptide containing q-g scissile bond as substrate for the in vitro screening of extracts. the authors isolated the novel benzoisochromanquinone ( )-thysanone ( fig. ; table iii ) from an extract of the fungus thysanophora penicilloides with potent hrv c-protease inhibitory activity. a continued screening revealed a pronounced hrv c-protease inhibitory activity for the extract of the chinese herb polygonum cuspidatum. by bioassay-guided fractionation -methoxystypandrone ( fig. ; table iii ), a naphthoquinone, with an ic value of . mm, was isolated from the plant material. the total syntheses of this natural compound and further analogues allowed for a structure-activity relationship, and particularly the comparison between activities of ortho-vs. para-quinones. to measure the selectivity of the compound series against cystein proteases other than hrv c-protease, the compounds were evaluated against papain. the simple , -phenanthraquinone ( fig. ; table iii ) was the most active compound of the series and showed an ic value of . mm with a distinctly higher degree of selectivity than -methoxystypandrone. a cpe reduction assay was applied for the identification of raoulic acid, a bicyclic c terpene acid ( fig. ; table iii ) isolated from raoulia australis (asteraceae). raoulic acid exerted an antiviral activity against coxsackie virus b , b , enterovirus , and hrv- and - with ic values in the submicromolar range. no activity was recorded against influenza a and b viruses. in the course of the systematic screening of microbial and natural products for anti-hrv activity, ishitsuka et al. identified , -dihydroxy- , , -trimethoxyflavone ( fig. ; table iii) from the leaves of the chinese medicinal plant agastache rugosa (lamiaceae) as natural compound with high activity against all picornaviruses except mengovirus. the authors synthesized the orally active , -diacetyloxy- , , -trimethoxyflavone and performed investigations in tissue cultures and in mice to determine the compound's mode of action. this was assumed to be the process of viral replication, thus located between viral uncoating and initiation of viral rna synthesis. the activity of flavan ( fig. ; table iii ) was discovered serendipitously during a screening program by using a plaque inhibition assay. based on these results, bauer et al. synthesized , -dichloroflavan (bw c, fig. ; table ii) , which revealed an activity against a number of hrv serotypes in the range between . and mm. as soon as an active lead compound is identified it is of utmost importance to scrutinize the derivatives' activity to (i) obtain insight into the chemical requirements mandatory for the focused biological activity, (ii) improve the compound's pharmacological profile in terms of potency, selectivity, cytotoxicity, etc., and to (iii) improve its bioavailability. the nonphenolic aporphine alkaloid glaucine is a prominent constituent of the aerial parts of gaucium flavum (papaveraveae). in a recently published study, spasova et al. investigated the antiviral potential of glaucine ( fig. ; table iii) , glaucine derivatives, and its semi-synthesized -aminoethylglaucine cinnamoyl-and hydroxycinnamoyl amides. beside the anti-oxidative potential of the newly synthesized compounds, they all exerted an antiviral activity against the replication of hrv- . the best anti-hrv activity was observed for oxoglaucine ( fig. ; table iii; ic table iii; ic mm, si . and ic mm, si , respectively). the early findings of the anti-hrv active naturally derived -methoxyflavone inspired chemists and pharmacologists in the synthesis and the pharmacological evaluation of derivatives thereof decorated with various substitution patterns. several reports published during the last two decades reviewed the findings of antiviral flavonoid research. , , by investigating the antiviral activity of a wide variety of naturally occurring flavonoids, tsuchiya et al. found chrysosplenol b and c ( fig. ; table iii ) contained in chrysosplenium plants, and axillarin ( fig. ; table iii ) as potent anti-hrv agents. based on their findings, the flavone skeleton decorated with a methoxy group in position and a -hydroxyl group revealed as mandatory for an anti-hrv activity. a series of antipicornaviral -hydroxy- -methoxyflavone derivatives was synthesized by de meyer et al. in order to establish a structure-activity relationship. thereby, different substitution patterns of the a-ring system with methyl, hydroxy, methoxy, halo, nitro, and amino was performed. their activity against polio and hrv was compared with those of naturally occurring flavonoids. further, the importance of the hydroxyl-group and of the -methoxyl-group was confirmed by investigation of different derivatives lacking these features. the results showed that -hydroxy- -methoxyflavones with a monosubstituted a-ring are less active than the corresponding compounds having a polysubstituted a-ring. within the tested series of compounds, , -dihydroxy- -methoxy- , -dimethylflavone emerged not only as noncytotoxic but also as most potent substance in both antiviral test systems. the lowest concentrations for this compound that protect % of the cells from cpe of hrv serotypes were in the range from . to . mg/ml. in contrast to quercetin, this flavone was also reported to have no mutagenic properties (measured up to . mg/plate) in a short-term microbial assay. the mechanism studies performed with -methoxyflavones have shown an interference with an early stage in the viral rna synthesis; no induction of resistance was observed. in contrast to this mode of action, the anti-hrv chalcones and flavans are reported to interact directly with specific sites on the viral capsid proteins, thereby preventing uncoating and the consequent liberation of viral rna. , , due to its anti-hrv potency, low toxicity, and promising bioavailability, dichloroflavan was evaluated for its protective efficacy against experimental hrv-infection in two clinical, double-blind, placebo-controlled trials. however, the drug candidate failed either when administered orally or intranasally. , unfortunately, till now no clinical trials evaluating the efficacy of -methoxyflavones in common cold have been performed. the common idea of all computational approaches is to extract knowledge from a more or less large set of data in order to make predictions of new events. within the lead discovery process, computational approaches, such as virtual screening, docking, quantitative structure-activity relationship, have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in drug research. the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. a number of books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts. [ ] [ ] [ ] [ ] in general, in silico methods can be divided into (i) ligand-based approaches, which rely on known active compounds. based on their physicochemical properties crucial for biological affinity, activities are predicted by extrapolation on not-yet tested substances, e.g. machine learning techniques and classical quantitative structure-activity relationship (qsar). ligand-based approaches are invaluable tools in cases where no structural information about the pharmacological targets is available. (ii) on the other hand, structurebased approaches use experimentally determined d structures of the targets, such as molecular docking or structure-based pharmacophore modeling for virtual screening. these methods allow for gaining insight into protein-ligand interactions at an experimentally determined (static) level (however not considering flexibility). a unique platform containing d coordinates of experimentally solved protein structures (by x-ray crystallography or nmr) is the protein data bank (pdb) currently comprising more than , structures of biomolecules and protein-ligand complexes. additionally, there are several pdb-related web services and tools, which enable to use the pdb-portal in a rich diversity of information services for students and scientists. particularly in the early stage of drug development, such as lead discovery and lead optimization, computational approaches allow for a target-oriented and rationalized proceeding, and thus may substantially help to maximize the success rate. a recently published review on the impact of computer-assisted approaches in antiviral research thoroughly describes underlying in silico techniques, and highlights the benefits of computational approaches for the discovery of antiviral lead structures. in anti-hrv research the capsid protein and the protease c revealed to be promising targets (as described before). inhibitors are assumed to have a major impact for the treatment of hrv-infections. additionally, these targets are structurally elucidated, and some potent ligands are known as well. these facts enable the performance of sensible computer-assisted approaches, both ligand-and structure-based. some studies using an in silico approach for the discovery of potential anti-hrv agents focusing on the mentioned targets will be reported in the following paragraphs. for compounds acting as potential hrv-capsid binders, some classical qsar and d qsar studies have been performed. while in classical qsar, the relationship between d calculated properties derived from chemical structures and measured biological activities are explored statistically, d qsar techniques are aimed at deriving a correlation and in turn activity prediction based on spatial arrangements of chemical properties and atoms. applying the d qsar technique comfa (comparative molecular field analysis) statistical models are derived, which are visualized in color-coded contours around the molecule. therein spots indicate where electrostatic properties and spatial arrangements are favorable for biological activity. in the studies of diana et al., artico et al., and verma et al., qsar techniques helped on one hand to analyze and rationalize the structural features of active compounds essential for the interaction to the hrv canyon's binding pocket, and on the other hand to search for new classes of capsid binders, thus to narrow the synthetic challenges for specific anti-hrv agents, respectively. [ ] [ ] [ ] [ ] [ ] in all these investigations hydrophobicity was found to be one of the most important determinants of substance activity. qsar combined with simplex representation of molecular structure was applied by kuz'min et al. based on the selectivity index (cc /ic ) and the hrv- inhibitory concentration of a set of [(biphenyloxy)propyl]isoxazole derivatives. on the basis of qsar analysis and computational design, three new isoxazoles with high activity prediction were selected and synthesized. they all revealed a strong coincidence between experimental and predicted anti-hrv activity and selectivity index. terminal benzene substituents with negative electrostatic potential and a molecule length of approximately . - . Å have been suggested as mandatory features within this chemical class for a hrv- inhibitory activity. hrv-serotypes show a high level of conservation at the protease c binding site; sequence alignment and secondary structure predictions suggested an overall architecture and mechanism of hrv- c proteases that correlates with cellular cystein-and serine proteases, such as chymotrypsin and trypsin. , the identity among c proteases from different families is however modest and provides space for the development of specific inhibitors for hrv- c protease. in a recently published review on selective inhibitors of picornavirus replication, de palma et al. summarized all currently known chemical structures acting as peptidic or nonpeptidic inhibitors of this viral target. in , reich et al., used the hrv c protease co-crystal structure information to rationalize the target-oriented synthesis of hrv c protease inhibitors from the class of substituted benzamides. activity data and subsequent crystallographic studies pointed out important requirements for the inhibition of the c protease. similarly, maugeri et al. used a structure-based approach, and performed docking studies based on the c protease crystal structure with a virtual library consisting of benzamide derivatives. quantum-mechanic calculation proposed substituents with most promising biological activities. this workflow guided the design and synthesis of substances virtually assumed to act as substrate analogues. synthesis of some of these compounds and biological testing confirmed the underlying hypothesis. quantitative molecular modeling studies were performed to better define and predict interactions between bicyclic -pyridone derivatives that showed to be irreversible inhibitors of the c protease. in these studies molecular mechanics simulations to evaluate chemical rate of covalent bond formation and free energy calculation combined with crystallographic studies were applied to explain the differences in activity of some irreversible peptidomimetic inhibitors. these data were used as a basis for further optimization of these compounds. , in a recent study performed by kuo et al. some , compounds were subjected to a high troughput screening in the search for novel inhibitors for both c and cl proteases from picornavirus and coronavirus, respectively. five nonpeptides were identified with ic values r mm against severe acute respiratory syndrome-coronavirus cl-protease; one molecule was found to additionally inhibit the c proteases of coxsackievirus, enterovirus, and rhinovirus. this compound (id ) contains a dihydropyrazole ring decorated with two phenyl groups and a lengthy n-butyl-benzimidazolylamino-toluene. it was used as starting point for the selection of further four analogs showing ic values in the range of . - mm against the tested viral proteases. by means of docking-based computer modeling, the authors tried to rationalize the binding discrepancies responsible for individual and common protease inhibitors, thus to provide a rational base for the development of nonpeptide multiple-function inhibitors against coronaviruses and piciornaviruses. in anti-hrv research, first application scenarios have been conducted using pharmacophore models. according to the official iupac definition by wermuth et al., a pharmacophore describes the d arrangement of steric and electronic features necessary to trigger or block a biological response. pharmacophores can be represented by three-dimensional chemical features, which include hydrogen bond donors and acceptors, aromatic rings, hydrophobic groups, as well as positive and negative ionisable moieties. additionally, the shape of ligands can be represented by shape features, which essentially describe the van der waals radii of the ligand atoms. the pharmacophore concept has proven to be successful, not only in rationalizing structure-activity relationships but also by its large impact in developing appropriate d-tools for efficient virtual screening. , steindl et al. elaborated ligand-and structure-based pharmacophore models implementing the essential feature of the covalent binding to the cysteine in the active site of the hrv- c protease. thus, the in silico approach focused on defining a new pharmacophore feature representing a target structure for nucleophilic addition in the ligands, which is a crucial step for protease inactivation. the generated hypotheses retrieved known c protease inhibitors in the virtual screening cycle, and proposed potential (unconfirmed) ligands of the d protease binding site from available databases. the viral capsid of several hrvs has been elucidated by crystallization and resolution of the d-structure. the hrv coat protein complexed with its highly active inhibitor win was used as starting point for the generation of structure-based pharmacophore models by . the models were used for virtual screening of a large commercially available d database. for final selection of virtual hits worth to be subjected to biological testing, docking studies and principal component analysis were performed. six candidates were tested for their ability to inhibit hrv serotype by multiple-cycle cpe inhibition assay. although all of them showed a certain antiviral potential, one longitudinal piperazine derivative inhibited the virus at a concentration below mm. some of the test candidates showed difficulties in the interpretation of experimental results due to their relatively high cytotoxicity and bad solubility. this circumstance asks for more cautious estimation of molecular properties for compound selection as stated by the authors. in a subsequently performed study, the best validated pharmacophore model was used for the identification of naturally derived hrv coat protein inhibitors. for virtual screening experiments the in-house generated d-database dios was used (as described before). based on the virtually predicted ligands and considering knowledge from traditional use, sesquiterpene umbelliferons from the gum resin of ferula sp., i.e. asafetida, were finally selected as most promising candidates. for biological evaluation, the antiviral activities of asafetida and its isolated constituents were assessed by an exploratory determination of the inhibition of the cpe induced by hrv serotypes a, , , and . the results revealed a dose-dependent and selective anti-hrv activity against serotype for asafetida and its virtually predicted constituents, farnesiferols b and c ( fig. ; table iii ; ic : . and . mm, respectively). to scrutinize the selectivity of these two compounds against hrv- in comparison to the other tested serotypes, the amino acid sequences of hrv- and hrv- vp were aligned. since all amino acid residues involved in ligand binding showed % match in both serotypes, the experimentally determined selectivity profile could not be explained by different binding pockets. the serotype alignment does however not reflect potential protein flexibility during binding, which might differ between the hrv serotypes. additionally, off-target effects could be a reason for the observed selectivity. in a recently performed virtual parallel screening approach, we tried to identify potential targets of human pathological relevance for constituents isolated and identified from the medicinal plant ruta graveolens. using the screening platform pipline pilot included in discovery studio, low-energy conformers of the identified molecules from r. graveolens were subjected to parallel screening. for this purpose, the inte:ligand pharmacophore model collection was used. it currently comprises . models covering unique pharmacological targets. based on the predicted ligand-target interactions, the authors focused on three biological targets, namely acetylcholinesterase, the hrv coat protein, and the cannabinoid receptor type . virtual hits and nonhits were assayed on their respective targets for a critical evaluation of the performed target-fishing approach. beside other predicted bioactivities, determination of their cpes on hrv- revealed the virtual hit arborinine ( fig. ; table iii; ic : . mm) and the nonpredicted hit , , -trimethoxycoumarin ( fig. ; table iii ; ic : . mm) as the most active anti-hrv constituents. it could be shown that the applied in silico strategy has the capacity of catalyzing drug discovery profoundly for all those diseases where molecular targets or molecular ligands are well defined to create reliable pharmacophore models. hrv, a prominent member of the picornaviridae family, infects humans more frequently than any other virus. infections with hrv mainly lead to upper respiratory diseases, such as the common cold, but may also cause more severe lower respiratory tract disorders. although the symptomology and severity of the common cold is relatively mild and the course of disease self-limiting, the socio-economic impact is tremendous in terms of recouping lost productivity due to sick leave. in the last decades, a number of anti-hrv agents from different origin-synthetics, natural compounds, biologicals, botanicals, and nutritionals-have been discovered. different concepts have been used as strategy for their discovery either starting from a phenomenological effect, e.g. empirical knowledge from folk medicine, or from a targetbased molecular level, e.g. icams, capsid binders, and hrv protease inhibitors. some of the outcomes revealed potent and promising activities that partly have been evaluated for the management of hrv-induced common colds in clinical trials mainly with sobering benefit. since the hrv infection is not life-threatening in most cases, a potential therapy has to be safe and effective with an almost unrecognizable level of side effects. these preconditions render the search for anti-hrv-agents into a high challenging endeavor and explains why no antiviral agent is approved for the prevention or treatment of hrv-infection until today, despite the significant efforts. moreover, the high variability of rhinoviruses suggests the need of more than one active principle covering different modes of action for an effective treatment. therefore, there is a high need for an ongoing search for new synthetic as well as natural compounds on a molecular level. the increasing knowledge about the hrv life cycle, gene, and protein sequence combined with the improved technologies in the field of experimental and computational methods have continuously enabled further insights into the spectacular world of hrv. only with this fundamental research, virtual screening approaches, such as qsar, pharmacophore-and docking-based screening cycles, or computational compound design, are applicable on a rational base. with the gained expertise from different disciplines and an adequate infrastructure for further research, it is encouraging to hope that discovery and clinical development efforts will continue in the search for agents that may treat or prevent the annoying common cold. picornavirales, a proposed order of positive-sense single-stranded rna viruses with a pseudo-t virion architecture global distribution of novel rhinovirus genotype genome-wide diversity and selective pressure in the human rhinovirus clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution comparison of classic and molecular approaches for the identification of untypeable enteroviruses molecular evolution of the human enteroviruses: correlation of serotype with vp sequence and application to picornavirus classification typing of human enteroviruses by partial sequencing of vp molecular relationships between human rhinovirus serotypes vp sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds molecular cloning and complete sequence determination of rna genome of human rhinovirus type evolutionary relationships within the human rhinovirus genus: comparison of serotypes , , and amino acid changes in proteins b and a mediate rhinovirus type growth in mouse cells the nucleotide sequence of human rhinovirus b: molecular relationships within the rhinovirus genus complete sequence of the rna genome of human rhinovirus , a clinically useful common cold virus belonging to the icam- receptor group human rhinovirus : complete nucleotide sequence and proteolytic processing signals in the capsid protein region the complete nucleotide sequence of a common cold virus: human rhinovirus new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus the common cold phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany acute respiratory illness in the community. frequency of illness and the agents involved sinusitis in the common cold viral etiology of common cold in children a case-control study of acute respiratory tract infection in general practice patients in the netherlands community-acquired pathogens associated with prolonged coughing in children: a prospective cohort study frequency and natural history of rhinovirus infections in adults during autumn viruses and bacteria in the etiology of the common cold the seasonality of rhinovirus infections and its implications for clinical recognition respiratory pathogens in children with and without respiratory symptoms virological and serological analysis of rhinovirus infections during the first two years of life in a cohort of children novel species of human rhinoviruses in acute otitis media spectrum of clinical illness in hospitalized patients with ''common cold'' virus infections respiratory viruses and severe lower respiratory tract complications in hospitalized patients respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection rhinovirus infection preferentially increases lower airway responsiveness in allergic subjects rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia association of respiratory picornaviruses with acute bronchiolitis in french infants chronic rhinoviral infection in lung transplant recipients risk factors for asthma and atopy interleukin- gene expression in acute virus-induced asthma community study of role of viral infections in exacerbations of asthma in - year old children asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus viral pathogens in acute exacerbations of chronic obstructive pulmonary disease respiratory viral infection in exacerbations of copd the role of respiratory infections in chronic obstructive pulmonary disease respiratory viral infections drive chemokine expression and exacerbate the asthmatic response respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease respiratory syncytial virus infection in infants admitted to paediatric intensive care units in london, and in their families effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis the role of respiratory viruses in cystic fibrosis novel human rhinoviruses and exacerbation of asthma in children a novel group of rhinoviruses is associated with asthma hospitalizations rhinovirus transmission: one if by air, two if by hand how contagious are common respiratory tract infections? environmental contamination with rhinovirus and transfer to fingers of healthy individuals by daily life activity aerosol transmission of rhinovirus colds hand-to-hand transmission of rhinovirus colds transmission of experimental rhinovirus infection by contaminated surfaces interruption of experimental rhinovirus transmission virucidal activity and cytotoxicity of the liposomal formulation of povidone-iodine virucidal hand treatments for prevention of rhinovirus infection localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization experimental rhinovirus infection in volunteers detection of rhinovirus rna in lower airway cells during experimentally induced infection relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds sites of rhinovirus recovery after point inoculation of the upper airway incubation periods of experimental rhinovirus infection and illness quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinoviruses infect the lower airways incidence and characteristics of viral community-acquired pneumonia in adults etiology of community-acquired pneumonia in hospitalized children improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections association between interleukin- concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds symptom profile of common colds in schoolaged children the microbial etiology and antimicrobial therapy of adults with acute community-acquired sinusitis: a fifteen-year experience at the university of virginia and review of other selected studies bacterial coinfections in children with viral wheezing amplified rhinovirus colds in atopic subjects experimental rhinovirus infection potentiates histamine release after antigen bronchoprovocation in allergic subjects lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects the effects of rhinovirus infections on allergic airway responses rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production the economic burden of non-influenzarelated viral respiratory tract infection in the united states rhinovirus chemotherapy excessive antibiotic use for acute respiratory infections in the united states influenza virus and rhinovirus-related otitis media: potential for antiviral intervention the viruses and their replication analysis of the structure of a common cold virus, human rhinovirus , refined at a resolution of . a the refined structure of human rhinovirus at . a resolution: implications for the viral life cycle crystal structure of human rhinovirus serotype a (hrv a) the structure of human rhinovirus structure of a human common cold virus and functional relationship to other picornaviruses structure of human rhinovirus serotype (hrv ) human rhinovirus at . a resolution the canyon hypothesis viral cell recognition and entry crystallographic and cryo em analysis of virion-receptor interactions a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses the minor receptor group of human rhinovirus (hrv) includes hrv and hrv , but the presence of a lysine in the vp hi loop is not sufficient for receptor binding members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus multiple receptors involved in human rhinovirus attachment to live cells structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor the cellular receptor to human rhinovirus binds around the -fold axis and not in the canyon: a structural view the major and minor group receptor families contain all but one human rhinovirus serotype human rhinovirus type infection via heparan sulfate is less efficient and strictly dependent on low endosomal ph human rhinovirus type variants use heparan sulfate proteoglycan for cell attachment internalization of human rhinovirus into hela and icam- -transfected bhk cells viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in icam- -negative cells rhinovirus-mediated endosomal release of transfection complexes major and minor receptor group human rhinoviruses penetrate from endosomes by different mechanisms cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type uncoating formation of rhinovirus-soluble icam- complexes and conformational changes in the virion conformational changes, plasma membrane penetration, and infection by human rhinovirus type : role of receptors and low ph uncoating of human rhinovirus serotype from late endosomes x-ray structure of a minor group human rhinovirus bound to a fragment of its cellular receptor protein the expression and purification of human rhinovirus protease c polypeptide a of human rhinovirus type : identification as a protease and characterization by mutational analysis the eif g-eif e complex is the target for direct cleavage by the rhinovirus a proteinase purification of two picornaviral a proteinases: interaction with eif- gamma and influence on in vitro translation a proteinases of coxsackie-and rhinovirus cleave peptides derived from eif- gamma via a common recognition motif conservation of amino acids in human rhinovirus c protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus c protease inhibitor fields virology rhinovirus infects primary human airway fibroblasts and induces a neutrophil chemokine and a permeability factor basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection resistance of differentiated human airway epithelium to infection by rhinovirus effect of isoflavans and isoflavenes on rhinovirus b and its replication in hela cells enantiomeric effects of homologues of disoxaril on the inhibitory activity against human rhinovirus- in vitro activity of win , a new broad-spectrum antipicornavirus drug two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity an orally bioavailable oxime ether capsid binder with potent activity against human rhinovirus in vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity novel [(biphenyloxy)propyl]-isoxazole derivatives for inhibition of human rhinovirus and coxsackievirus b replication structure-based virtual screening for the discovery of natural inhibitors for human rhinovirus coat protein in vitro resistance study of rupintrivir, a novel inhibitor of human rhinovirus c protease establishment of a mouse model for human rhinovirus infection human rhinovirus b exposure induces phosphatidylinositol -kinase-dependent airway inflammation in mice mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation prophylactic activity of intranasal enviroxime against experimentally induced rhinovirus type infection intranasal interferon-alpha treatment of experimental rhinoviral colds topical enviroxime against rhinovirus infection therapeutic activity of enviroxime against rhinovirus infection in volunteers efficacy of oral win for prophylaxis of experimental rhinovirus infection ineffectiveness of echinacea for prevention of experimental rhinovirus colds current status of anti-picornavirus therapies selective inhibitors of picornavirus replication inhibitors of c cysteine proteinases from picornaviridae isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses an antibody fragment from a phage display library competes for ligand binding to the low density lipoprotein receptor family and inhibits rhinovirus infection modification of experimental rhinovirus colds by receptor blockade viral receptor blockage by multivalent recombinant antibody fusion proteins: inhibiting human rhinovirus (hrv) infection with cfy comparative antirhinoviral activities of soluble intercellular adhesion molecule- (sicam- ) and chimeric icam- /immunoglobulin a molecule a soluble form of intercellular adhesion molecule- inhibits rhinovirus infection spectrum of activity of soluble intercellular adhesion molecule- against rhinovirus reference strains and field isolates soluble ldl minireceptors. minimal structure requirements for recognition of minor group human rhinovirus neutralization of a common cold virus by concatemers of the third ligand binding module of the vldl-receptor strongly depends on the number of modules rhinovirus-stabilizing activity of artificial vldl-receptor variants defines a new mechanism for virus neutralization by soluble receptors in vitro studies of the antirhinovirus activity of soluble intercellular adhesion molecule- mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of icam- efficient neutralization and disruption of rhinovirus by chimeric icam- /immunoglobulin molecules recombinant soluble low density lipoprotein receptor fragment inhibits minor group rhinovirus infection in vitro prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule- efficacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection: a randomized clinical trial inhibitors of picornavirus replication the structure of antiviral agents that inhibit uncoating when complexed with viral capsids structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds structural analysis of antiviral agents that interact with the capsid of human rhinoviruses conformational change in the floor of the human rhinovirus canyon blocks adsorption to hela cell receptors the site of attachment in human rhinovirus for antiviral agents that inhibit uncoating a comparison of the anti-rhinoviral drug binding pocket in hrv and hrv a analysis of three structurally related antiviral compounds in complex with human rhinovirus antiviral capsid-binding compounds can inhibit the adsorption of minor receptor rhinoviruses in vitro activity of pleconaril and ag against selected serotypes and clinical isolates of human rhinoviruses activity of pleconaril against enteroviruses susceptibility of coxsackievirus b laboratory strains and clinical isolates to the capsid function inhibitor pleconaril: antiviral studies with virus chimeras demonstrate the crucial role of amino acid in treatment in vitro activity of r , a new antirhinovirus compound in vitro activity of pirodavir (r ), a substituted phenoxy-pyridazinamine with broad-spectrum antipicornaviral activity quantitative structure-activity relationship studies of [(biphenyloxy)-propyl]isoxazole derivatives. inhibitors of human rhinovirus replication sch : a potent, broad-spectrum, antienterovirus compound antipicornavirus activity of sch and analogs: in vitro and in vivo studies sch : a picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating direct and specific inactivation of rhinovirus by chalcone ro - comparative studies on the antirhinovirus activity and the mode of action of the rhinovirus capsid-binding agents, chalcone amides , -dichloroflavan (bw c), a new anti-rhinovirus compound , -dihydro- -phenyl- h-pyrano[ , -b]pyridines with potent antirhinovirus activity antipicornavirus activity of substituted phenoxybenzenes and phenoxypyridines activity of -( , -dichlorophenoxy)- -nitrobenzonitrile (mdl- ) against picornaviruses in vitro application of crystallography to the design of antiviral agents clinical activity of pleconaril in an experimentally induced coxsackievirus a respiratory infection oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: efficacy and tolerability in phase ii clinical trials efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses the effect of oral pleconaril on hepatic cytochrome p a activity in healthy adults using intravenous midazolam as a probe safety and efficacy of intranasal pirodavir (r ) in experimental rhinovirus infection inhibition of polyprotein processing and rna replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease a through s-nitrosylation: a protective mechanism against enteroviral cardiomyopathy an antiviral mechanism of nitric oxide: inhibition of a viral protease nitric oxide inhibition of coxsackievirus replication in vitro nitric oxide donors inhibit the coxsackievirus b proteinases a and c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice effect of nitric oxide on rhinovirus replication and virus-induced interleukin- elaboration inhibition of proteolytic activity of poliovirus and rhinovirus a proteinases by elastase-specific inhibitors an antiviral peptide inhibitor that is active against picornavirus a proteinases but not cellular caspases antiviral activity of caspase inhibitors: effect on picornaviral a proteinase dual inhibition of human rhinovirus a and c proteases by homophthalimides structure of human rhinovirus c protease reveals a trypsin-like polypeptide fold, rna-binding site, and means for cleaving precursor polyprotein individual and common inhibitors of coronavirus and picornavirus main proteases structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes new anti-viral drugs for the treatment of the common cold structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . michael acceptor structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . peptide structure-activity studies structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . structure-activity studies of orally bioavailable, -pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . structure-activity studies of ketomethylene-containing peptidomimetics structurebased design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . pharmacological optimization of orally bioavailable -pyridone-containing peptidomimetics structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . incorporation of p lactam moieties as l-glutamine replacements in vitro antiviral activity of ag , a potent inhibitor of human rhinovirus c protease inhibition of human rhinovirus-induced cytokine production by ag , a human rhinovirus c protease inhibitor pharmacokinetics and safety of an antirhinoviral agent, ruprintrivir, in healthy volunteers phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray -percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus c protease gaining target access for deoxyribozymes a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism mode of action of -furylmercury chloride, an antirhinovirus compound methoxyflavones as potent inhibitors of viral-induced block of cell synthesis antiviral effects of pyrrolidine dithiocarbamate on human rhinoviruses broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at ctp synthetase in vitro antiviral activity of ribavirin against picornaviruses another ten stories in antiviral drug discovery (part c): ''old'' and ''new'' antivirals, strategies, and perspectives the broadspectrum antiviral ribonucleoside ribavirin is an rna virus mutagen ribavirin: recent insights into antiviral mechanisms of action inhibition of rhinovirus replication in in organ culture by a potential antiviral drug synthesis of syn and anti isomers of -[[(hydroxyimino)phenyl]methyl]- -[( -methylethyl)sulfonyl]- h-benzimidaz ol- -amine. inhibitors of rhinovirus multiplication comparative studies on the modes of action of the antirhinovirus agents ro - , ro - , rmi- , , , -dichloroflavan, and enviroxime the antiviral compound enviroxime targets the a coding region of rhinovirus and poliovirus sequence determinants of a-mediated resistance to enviroxime in rhinoviruses and enteroviruses evidence that enviroxime targets multiple components of the rhinovirus replication complex controlled trial of enviroxime against natural rhinovirus infections in a community the activity of enviroxime against rhinovirus infection in man activity against rhinoviruses, toxicity, and delivery in aerosol of enviroxime in liposomes -amino- -substituted- -[(e)- -phenyl- -(n-methylcarbamoyl)vinyl]imid azo[ , -a]pyridines as a novel class of inhibitors of human rhinovirus: stereospecific synthesis and antiviral activity short synthesis and anti-rhinoviral activity of imidazo[ , -a]pyridines: the effect of acyl groups at -position synthesis, antiviral activity, and biological properties of vinylacetylene analogs of enviroxime synthesis and antiviral activity of c analogs of enviroxime: an exploration of the role of critical functionality sensitivity of rhinoviruses to human leukocyte and fibroblast interferons cell and virus sensitivity studies with recombinant human alpha interferons comparative susceptibility of respiratory viruses to recombinant interferons-alpha b and -beta intranasal interferon-alpha for prevention of natural rhinovirus colds intranasal interferon alpha for prevention of rhinovirus infection and illness interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of huifn-alpha efficacy and tolerance of intranasally applied recombinant leukocyte a interferon in normal volunteers intranasally applied recombinant leukocyte a interferon in normal volunteers. ii. determination of minimal effective and tolerable dose recombinant human interferon-gamma as prophylaxis against rhinovirus colds in volunteers ineffectiveness of postexposure prophylaxis of rhinovirus infection with low-dose intranasal alpha b interferon in families a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection synergism between anti-rhinovirus antivirals: various human interferons and a number of synthetic compounds failure to demonstrate synergy between interferon-alpha and a synthetic antiviral, enviroxime, in rhinovirus infections in volunteers property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry a new rapid and effective chemistry space filter in recognizing a druglike database rational selection of structurally diverse natural product scaffolds with favorable adme properties for drug discovery natural products as sources of new drugs over the last years ethnomedicinal antivirals: scope and opportunity plant substances as antiviral agents: an update plant substances as antiviral agents host defense function of the airway epithelium in health and disease: clinical background herb sales down percent in mainstream market echinacea for preventing and treating the common cold echinacea for preventing and treating the common cold. the cochrane database of systematic reviews evaluation of echinacea for the prevention and treatment of the common cold: a meta-analysis echinacea in the prevention of induced rhinovirus colds: a meta-analysis the role of alkamides as an active principle of echinacea alkylamides from echinacea are a new class of cannabinomimetics: cannabinoid type receptordependent and -independent immunomodulatory effects synergistic anti-oxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from echinacea purpurea on in vitro oxidation of human low-density lipoproteins echinacea extracts modulate the pattern of chemokine and cytokine secretion in rhinovirus-infected and uninfected epithelial cells modulation of immune response gene expression by echinacea extracts: results of a gene array analysis preventing the common cold with a garlic supplement: a double-blind, placebocontrolled survey in vitro virucidal effects of allium sativum (garlic) extract and compounds enhancement of in vitro human immune function by allium sativum l. (garlic) fractions efficacy of cold-fx in the prevention of respiratory symptoms in community-dwelling adults: a randomized, doubleblinded, placebo controlled trial efficacy of an extract of north american ginseng containing poly-furanosyl-pyranosyl-saccharides for preventing upper respiratory tract infections: a randomized controlled trial safety and tolerability of north american ginseng extract in the treatment of pediatric upper respiratory tract infection: a phase ii randomized, controlled trial of dosing schedules inhibitory effect of ginsenoside rg and its derivative ginsenoside rg - h on no production and lymphocyte proliferation effect of cvt-e (cold-fx) versus a ginsenoside extract on systemic and gut-associated immune function inhibitory effect of ginseng polysaccharides on rotavirus infection antiviral activity of dammarane saponins against herpes simplex virus i effect of a traditional japanese herbal medicine, hochu-ekki-to (bu-zhong-yi-qi tang), on immunity in elderly persons effects of five kampohozais on the mitogenic activity of lipopolysaccharide, concanavalin a, phorbol myristate acetate and phytohemagglutinin in vivo immunological restoration and anti-tumor effect by japanese herbal medicine in aged mice antiinflammatory effects of bu-zhong-yi-qi-tang in patients with perennial allergic rhinitis hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells efficacy of a pelargonium sidoides preparation in patients with the common cold: a randomized, double blind, placebo-controlled clinical trial kern winfried v. pelargonium sidoides extract for acute respiratory tract infections traditionally used pelargonium species: chemistry and biological activity of umckaloabo extracts and their constituents aqueous ethanolic extract of the roots of pelargonium sidoides-new scientific evidence for an old anti-infective phytopharmaceutical efficacy of an aqueous pelargonium sidoides extract against herpesvirus protective effect of a natural carrageenan on genital herpes simplex virus infection in mice polysaccharides as antiviral agents: antiviral activity of carrageenan iota-carrageenan is a potent inhibitor of rhinovirus infection recommendations for reporting randomized controlled trials of herbal interventions: explanation and elaboration ethnobotany and the search for new drugs: ciba foundation symposium ethnobotany and natural products: the search for new molecules, new treatments of old diseases or a better understanding of indigenous cultures? how scientific is the science in ethnopharmacology? historical perspectives and epistemological problems pedanius dioscorides of anazarbus-de materia medica sesquiterpene coumarins from ferula assafoetida l antiviral flavonoid from pterocaulon sphacelatum, an australian aboriginal medicine in silico target fishing for rationalized ligand discovery exemplified on constituents of ruta graveolens l antiviral activity of natural occurring flavonoids in vitro antiviral agents from higher plants and example of structure-activity relationship of -methoxyflavones -hydroxy- -methoxyflavones with potent antipicornavirus activity biodiversity: a continuing source of novel drug leads structure and stereochemistry of thysanone: a novel human rhinovirus c-protease inhibitor from thysanophora penicilloides discovery, total synthesis, hrv c-protease inhibitory activity, and structure-activity relationships of -methoxystypandrone and its analogues antiviral activity of raoulic acid from raoulia australis against picornaviruses antipicornavirus flavone ro - cinnamoyl-and hydroxycinnamoyl amides of glaucine and their antioxidative and antiviral activities antiviral activity of flavones and flavans present status and prospects of flavonoids as antiviral agents inhibition of an early stage of rhinovirus replication by dichloroflavan (bw c) effect of dichloroflavan (bw c) on the stability and uncoating of rhinovirus type b failure of intranasally administered , -dichloroflavan to protect against rhinovirus infection in man failure of oral , -dichloroflavan to protect against rhinovirus infection in man neural networks as data mining tools in drug design chemoinformatics and drug discovery methods and principles in medicinal chemistry the impact of informatics and computational chemistry on synthesis and screening enhancing drug discovery through in silico screening: strategies to increase true positives retrieval rates neural networks for chemists the protein data bank the protein data bank (pdb), its related services and software tools as key components for in silico guided drug discovery development of antiviral agents using molecular modeling and virtual screening techniques comparative molecular field analysis (comfa). . effect of shape on binding of steroids to carrier proteins investigation on qsar and binding mode of a new class of human rhinovirus- inhibitors by comfa and docking experiments synthesis and structure-activity studies of some disubstituted phenylisoxazoles against human picornavirus comfa analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus- dihydro- -oxazolyl)phenoxy]alkyl]- -methylisoxazoles: inhibitors of picornavirus uncoating understanding human rhinovirus infections in terms of qsar site-directed mutagenesis suggests close functional relationship between a human rhinovirus c cysteine protease and cellular trypsin-like serine proteases the picornaviral c proteinases: cysteine nucleophiles in serine proteinase folds substituted benzamide inhibitors of human rhinovirus c protease: structure-based design, synthesis, and biological evaluation structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus c protease inhibitors. . structure-activity studies of orally bioavailable, -pyridone-containing peptidomimetics glossary of terms used in medicinal chemistry (iupac recommendations ) methods and principles in medicinal chemistry human rhinovirus c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening docking versus pharmacophore model generation: a comparison of highthroughput virtual screening strategies for the search of human rhinovirus coat protein inhibitors ca: accelrys. . discovery studio rollinger extended her studies to the fields of phytochemistry, ethnopharmacology, molecular modelling and its application in natural product science completing the habilitation thesis entitled ''the search for bioactive natural products rollinger is a project leader and an associate in various national projects, as well as executive guest editor for current pharmaceutical design. she has been awarded the phoenix science award ( ) and received several additional awards until she was a postdoctoral research fellow at the institute of immunology, university marburg, where she studied picornavirusimmune cell interactions and the virus-induced cytokine induction. thereafter, she was a postdoctoral fellow at the institute of virology and antiviral therapy key: cord- -skrye w authors: hai, le thanh; bich, vu thi ngoc; ngai, le kien; diep, nguyen thi ngoc; phuc, phan huu; hung, viet pham; taylor, walter r.; horby, peter; liem, nguyen thanh; wertheim, heiman f.l. title: fatal respiratory infections associated with rhinovirus outbreak, vietnam date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: skrye w during an outbreak of severe acute respiratory infections in orphanages, vietnam, / hospitalized children died. all hospitalized children and / children from outbreak orphanages tested positive for rhinovirus versus / control children (p = . ). outbreak rhinoviruses formed a distinct genetic cluster. human rhinovirus is an underappreciated cause of severe pneumonia in vulnerable groups. during an outbreak of severe acute respiratory infections in orphanages, vietnam, / hospitalized children died. all hospitalized children and / children from outbreak orphanages tested positive for rhinovirus versus / control children (p = . ). outbreak rhinoviruses formed a distinct genetic cluster. human rhinovirus is an underappreciated cause of severe pneumonia in vulnerable groups. t he world health organization estimates that ≈ million children die each year from acute respiratory tract infection (ari), and most live in developing countries ( ) . human rhinovirus (hrv), a common cause of mild upper respiratory tract infections, may also cause severe ari in children. we report on an outbreak of severe ari caused by hrv in children living in orphanages in vietnam. during december -february , twelve infants < months of age with severe ari infection were admitted to hospitals in hanoi. because all infants lived in orphanages in hanoi, the national hospital of pediatrics (nhp) initiated an outbreak investigation. data on demographic characteristics, clinical features, and outcomes were collected for the infants. researchers visited of the outbreak orphanages and control orphanage ( km apart, no severe ari observed). patient histories were obtained from orphanage staff; all infants were examined, and nasal and pharyngeal swab specimens were collected. respiratory specimens were tested by bacterial culture and multiplex reverse transcription pcr (rt-pcr, seeplex rv kit; seegene, seoul, south korea) for the following respiratory viruses: infl uenza a/b viruses, respiratory syncytial virus (rsv), rhinovirus, coronavirus (oc /hku and e/nl ), adenovirus, parainfl uenzavirus, and human metapneumovirus ( ) . this outbreak investigation was approved by the scientifi c committee of nhp. twelve patients with severe ari were admitted to the nhp intensive care unit over days during the cool months of december -february (temperature °c- °c). the hospitalized infants ( female) were -to -months-old. most ( / ) were from orphanage, which was selected for investigation. two hospitalized children had a known underlying condition: congenital hypothyroidism (n = ) and hiv infection (n = ). seven infants were underweight (sex-specifi c weight-for-age; z score < sds). all exhibited cough, coryza, wheezing, and dyspnea, and ( %) had a documented fever. acute respiratory distress syndrome developed in all a mean of . ( % ci ± days) days after onset. mean pressure of arterial oxygen/fractional inspired oxygen ratio was . ( % ci ± . , range . - . ). chest radiographs showed extensive bilateral infi ltrates. blood cultures for bacteria were negative. despite mechanical ventilation and administration of intravenous broad-spectrum antimicrobial drugs, patients died and patients recovered ( were lost to follow up). hrv was detected by rt-pcr in all infants; patients were co-infected with rsv and adenovirus (table) . in addition, bronchoalveolar lavage specimen from patient was hrv positive. the outbreak orphanage was visited within week of outbreak detection. the visit revealed that several other infants had been hospitalized elsewhere, but we could not obtain detailed data about them. we tested nasal-pharyngeal swab specimens (pooled) from all infants ( % < months of age) living in the outbreak orphanage and nasal swab specimens from the youngest children ( . % < months of age) at the control orphanage within weeks of the outbreak. in both orphanages, - children lived in room and shared the same bed, wiping cloths, basic utensils such as cups, and clothing. most children in the outbreak orphanage were female ( / [ %]), compared with % ( / ) in the control orphanage. of children whose specimens were tested by rt-pcr, % ( / ) of the infants from the outbreak orphanage had at least symptom of respiratory tract infection compared with ( %) of infants at the control orphanage. among children from the outbreak orphanage (both hospitalized and nonhospitalized [n = ]), a single pathogen was identifi ed in ( . %) infants and > pathogens were found in ( . %) children (table) . the most frequently detected pathogen was hrv (n = ). in the control orphanage, ( . %) children tested positive for hrv, and children were infected with pathogens. only nonpathogenic bacteria were cultured from the respiratory specimens (data not shown). because hrv was the predominant pathogen detected, we genotyped all hrv isolates directly from the specimens using a molecular typing assay based on phylogenetic comparisons of a -bp variable sequence (p -p ) in the ′-noncoding region with homologous sequences of the known serotypes ( , ) . we were able to sequence the p -p fragment of hrv genome from specimens positive for hrv: were from hospitalized patients ( r and r) (figure) , were from the outbreak orphanage (identifi cation numbers starting with da), and were from the control orphanage (identifi cation numbers starting with bavi; figure) . the p -p phylogenetic tree showed that sequences obtained from isolates from the hospitalized infants were closely related to a subclade of cluster a sequences from nonhospitalized infants at the outbreak orphanage (figure) . hrvs from the control orphanage are also part of the a cluster but formed a distinct subcluster. several children (not hospitalized) from the outbreak orphanage were also infected with hrvs from the c cluster. no children had b cluster strains. we found that hrv was the main pathogen detected in an outbreak of severe ari in children living in an orphanage in vietnam. our fi ndings support recent studies showing that hrv may be associated with severe respiratory tract infection in infants and children ( ) ( ) ( ) . a study in central vietnam also showed that hrv is a notable cause of ari in children in vietnam ( ) . because we detected rhinovirus in all hospitalized infants, we believe that hrv was the main causal agent in this outbreak, although hospitalized infants were co-infected with rsv, an unambiguous respiratory pathogen. a deep lung specimen from hospitalized infant was also positive for hrv, which supports a causal relationship. furthermore, the outbreak orphanage had signifi cantly more hrv-positive patients than did the control orphanage, and sequence analysis showed that the outbreak isolates formed a distinct cluster, which also supports a causal role for hrv. in addition, we found that several infants from the outbreak orphanages were infected with hrv strains belonging to the c cluster, according to p -p sequence analysis. hrv-c has been associated with more severe infections and circulates worldwide ( , ( ) ( ) ( ) . we may have missed the hrv-c strains in the clinical case-patients, because we were only able to sequence the virus from samples from patients. we had to sequence directly from the specimens because we had no viral culture facility at the time of the outbreak. phylogenetic analysis of p -p sequences can distinguish hrv-b rhinoviruses, but it is limited in distinguishing hrv-a and hrv-c. full hrv sequence analysis would be able to provide this detail, but it was not feasible for this investigation. this outbreak investigation has some limitations. only a small number of controls were selected from a single orphanage, and the controls were sampled later than the hospitalized patients and children from the outbreak orphanage. these limitations may have led to underdiagnosis of hrv in controls. bacterial co-infection cannot be ruled out as a cause of more severe infection in the hospitalized infants because most were receiving antimicrobial drugs at the time respiratory specimens were collected. viral respiratory infections can be more severe in malnourished infants, which was likely the case for the hospitalized infants in this outbreak. in this study, co-infections ( . %) with other respiratory viruses were detected at a similar rate as in another study in central vietnam ( . %) ( ) . this fi nding is also consistent with previous work indicating that hrv infections can occur with other respiratory viruses and lead to more severe disease ( , ) . this outbreak illustrates that hrvs can cause severe pneumonia, leading to acute respiratory distress syndrome in young, vulnerable infants. hrv remains an underappreciated cause of severe pneumonia in vulnerable groups. children: reducing mortality. world health organization factsheet no multipathogen infections in hospitalized children with acute respiratory infections a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality serious respiratory illness associated with rhinovirus infection in a pediatric population global distribution of novel rhinovirus genotype viral pathogens associated with acute respiratory infections in central vietnamese children sequence analysis of human rhinoviruses in the rna-dependent rna polymerase coding region reveals large within-species variation human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children genotyping of human rhinovirus (hrv) from outbreak and control specimens by p -p sequence analysis nucleotide distances were analyzed with dnadist, the neighbor-joining tree of bioedit package. the consensus tree was visualized by treeview v the opinions expressed by authors contributing to this journal do not necessarily refl ect the opinions of the centers for disease control and prevention or the institutions with which the authors are affi liated we thank the hospital and orphanage staff for their cooperation in this outbreak investigation. key: cord- - kiu pb authors: akinloye, oluwabukola m.; rönkkö, esa; savolainen-kopra, carita; ziegler, thedi; iwalokun, bamidele a.; deji-agboola, mope a.; oluwadun, afolabi; roivainen, merja; adu, festus d.; hovi, tapani title: specific viruses detected in nigerian children in association with acute respiratory disease date: - - journal: j trop med doi: . / / sha: doc_id: cord_uid: kiu pb occurrence of different viruses in acute respiratory tract infections of nigerian children was examined. respiratory swabs were collected from children referred to hospital clinics because of acute respiratory symptoms from february through may . validated real-time rt-pcr techniques revealed nucleic acids of at least one virus group in specimens ( %). human rhinoviruses and parainfluenza viruses were present each in one third of the children. adenoviruses, enteroviruses, human metapneumovirus, human bocavirus, and influenza c virus were also relatively common. possibly due to their seasonal occurrence, influenza a and b virus, and respiratory syncytial virus were detected rarely. we conclude that all major groups of respiratory tract viruses are causing illness in nigerian children. wide range of viruses is known to be associated with respiratory disease in humans. adenoviruses, coronaviruses, human enteroviruses (hev), human rhinoviruses (hrv), influenza viruses, parainfluenza viruses (piv), and respiratory syncytial viruses (rsv) are well-known causes of acute respiratory tract infections (arti) in both industrialized and developing countries. over the last decade, modern molecular techniques have led to the discovery of several previously unknown respiratory tract viruses, including human metapneumovirus (hmpv) [ ] , two new human coronavirus types [ , ] , human bocavirus (hbov) [ ] , and two new human polyomaviruses [ , ] . the significance of these novel viruses has been reviewed recently [ , ] . it is widely accepted that common cold is almost always caused by viruses, most frequently by hrv [ ] , and viral infections are considered to contribute to the generation of complications of common cold, such as acute otitis media and sinusitis. moreover, different viruses, including influenza viruses and rsv, are also frequently detected in samples obtained from patients with lower respiratory tract infection (lrti), either alone or together with pathogenic bacteria. several recent reports, including some from africa, suggest viruses as potential etiologic agents in pneumonia in children [ ] [ ] [ ] [ ] , or exacerbations of asthma [ ] [ ] [ ] . several studies underscore the importance of respiratory tract viruses in nigerian patients, but these studies were carried out before the introduction of modern molecular diagnostic techniques [ , [ ] [ ] [ ] . the present study was journal of tropical medicine < - - - - > noage total * our hev test can react with some hrv strains. "total" shows the number of all specimens testing positive in the hev assay, "hrv-" those where the hrv test was negative ("true hev positives"). designed to identify viral agents associated with respiratory infections among young children in nigeria using modern, validated molecular techniques. we wanted to explore the presence of different virus groups, including some of the newer ones detected by only molecular techniques. no. cs , copan diagnostics inc., murrieta, calif usa) into the nostril to a depth of - cm, and retracting it with a rotating motion, in order to trap epithelial cells in the swab. with a second swab, a throat specimen was collected by rubbing the tonsils and the posterior wall of the pharynx. the swabs were then placed in a vial containing ml of rnalater solution (rnalater tissue collection, applied biosystems, espoo, finland). the specimens were transported to the laboratory on the same day in an ice pack and stored at − • c until further processing. viral nucleic acids were extracted from µl of sample using a commercial kit (rneasy mini kit, qiagen, hilden, germany), and viral rna was reverse transcribed into cdna with random hexamer primers (roche, mannheim, germany) and revertaid reverse transcriptase enzyme (fermentas, st. leon-rot, germany) following the manufacturers' instructions. for the detection of influenza a and b viruses, rsv, piv , , and , and cdna were amplified in two separate real-time multiplex pcrs [ ] with minor modifications. other, slightly modified real-time pcrs were used for the detection of influenza a subtypes h and h (r. fouchier, rotterdam, the netherlands, personal communication), influenza c (l. p. nielsen, copenhagen, denmark, personal communication), bocavirus [ ] , metapneumovirus [ ] , and adenovirus [ ] . real-time rt-pcr for the detection of hrv was performed as described [ ] and of enteroviruses (hev) according to the method by centers for disease control and prevention, atlanta, usa (w.a. nix and d.r. kilpatrick, personal communication). we know that the hev test is not % specific but also reacts with some hrv strains (savolainen-kopra et al., unpublished results). therefore, specimens yielding a positive result in the hev test were divided into two groups, and a designated "true hev" result was scored only for those hev-positive specimens that were negative in the hrv test. more than half of the patients ( / , . %) were less than one year of age, and almost one half of the remaining children, whose age was recorded ( / , . %), were under two years of age (table ) . respiratory tract viruses were detected in ( %) of the children. the overall rate of virus positivity was similar in children less than year of age ( / , . %), between and years ( / , . %), and above years of age ( / , . %) and slightly lower among the % of children whose age was not recorded ( / , . %). the most frequently detected virus groups, both found in about one third of the children each, were hrv and piv, with type of piv being the most prevalent serotype in the latter group (table ) . adenoviruses, influenza virus c, hmpv, and hbov were also found in considerable numbers. influenza virus types a and b and rsv were detected less frequently. seven specimens tested positive for hev and negative for hrv ("true hev"), whereas other additional specimens yielded a positive result by the hev and the hrv test. while the overall proportion of virus-positive specimens was similar in children aged under or over two years, as well as in the group with unrecorded age, all adenovirus, influenza virus a, rsv, and all but one hbov and "true hev" detections were in the youngest age group (table ) . altogether virus findings were obtained from the children, if only those hev-positive results were accounted where the same specimen was negative in the hrv test ("true hev"). the number of findings was if all positive test results for hev were included. twenty-nine specimens contained two different viruses and another two specimens three different viruses. no obvious pattern could be seen in the mutual associations of two or three viruses (table ) . however, there were more multiple infections in children older than years than in the younger than years group ( % versus % of children, p = . ( -sample t-test for equality of proportions), data not shown. this study revealed that all major respiratory virus groups tested for can be detected in nigerian children with respiratory tract infection. all viruses investigated, including the more recently discovered hmpv and hbov, were present in at least some of the specimens. this wide variety is somewhat surprising because the specimens have been collected only during a four-month period, mostly in the dry season of the year. it is known from the previous studies that circulation of some viruses is at a low level during the dry season [ ] [ ] [ ] . seventy-seven percent of the children harboured at least one viral pathogen in their samples. this figure is relatively high taking in account that we did not have the possibility to test the specimens for human coronaviruses, and comparable to [ ] or higher than in some recent similar studies performed elsewhere [ ] [ ] [ ] . human rhinoviruses and piv were the two most frequently detected virus groups. simultaneous infections with two or even three viruses were also found, similar to observations by others in comparable studies [ , ] . in individual cases, a demonstration of viral nucleic acids in a nasal or throat swab does not necessarily prove an etiological role of this pathogen in the concurrent disease as positive test results have also been obtained from a proportion of healthy individuals [ ] . etiological diagnosis of lrti is especially difficult without invasive procedures, but when searched for, for example, rhinovirus has been found in alveolar lavage specimens from pneumonia patients [ ] establishing the role of hrv as a significant respiratory pathogen beyond being the major causative agent of common cold [ ] . previous studies on respiratory tract viruses in nigeria have not tested for the presence of rhinoviruses. in our study, hrv accounted for % of the virus findings in the study population reiterating the observations done elsewhere [ , , , ] . our test for the related hev revealed a positive result in about % of the specimens, but two thirds of these specimens also tested positive for hrv. because of the well-established cross-reactions [ ] , these doublereactive specimens were recorded as hrv, and only those hev-positive specimens that were hrv negative were scored as "true hev positives." however, we cannot exclude concomitant infection with both hev and hrv in these double-reactive cases. a high incidence of parainfluenza viruses was found in our study population with piv being the most frequent of the three serotypes tested for. interestingly, piv has also been one major agent in some previous studies done in nigeria [ , ] and one in cambodia [ ] . respiratory syncytial virus was a rare finding in our study, likely due to the seasonal occurrence of this virus. our specimens were collected in february-may which has been a low season for rsv in nigeria in previous studies [ , ] . little was known about the epidemiology of influenza viruses in west africa until very recently. a report from senegal [ ] published, while this manuscript was in preparation, showed a september-october peak of influenza a and rsv. hence, seasonality might have again been contributing to the paucity of influenza virus a and b findings among our patients. the two influenza a viruses further characterized were of the h n subtype, that is, the same subtype that was predominant during the - influenza season (http://apps.who.int/globalatlas/dataquery/default.asp). in contrast, influenza virus type c was found surprisingly often. with an incidence of almost %, influenza c was as common as adenoviruses in this study. double or triple infections were detected in % of the virus-positive children, which is comparable to that observed by others [ , ] . we do not have any explanation for the observed relatively higher frequency in the older children. one could speculate about increasing number of contacts by age in the community, but we did not collect information enabling further analysis of this possibility. in conclusion, the findings of this study emphasize the presence of all major groups of viruses in association with respiratory illnesses in young children of west africa. rhinoviruses and parainfluenza viruses were the most prevalent virus groups while influenza a and b viruses, as well rsv were rarely detected, possibly due to low season of these viruses during the time of sample collection. the authors declare no conflict of interests. a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples a newly reported human polyomavirus, ki virus, is present in the respiratory tract of australian children identification of a novel polyomavirus from patients with acute respiratory tract infections novel respiratory virus infections in children, brazil review of new and newly discovered respiratory tract viruses in children viral etiology of common cold in children, finland viral etiology of severe pneumonia among kenyan infants and children single and multiple viral infections in lower respiratory tract infection the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus microbial inciters of acute asthma in urban nigerian children rhinovirus and the lower respiratory tract persistence of rhinovirus rna after asthma exacerbation in children viral pathogens of acute lower respiratory infections in preschool nigerian children and clinical implications of multiple microbial identifications viral respiratory diseases in nigeria: a serological survey, ii. complement fixing antobody levels of adenoviruses, respiratory syncytial virus, psittacosis virus viral respiratory diseases in nigeria: a serological survey. i. complement fixing antibody levels of influenza a, b and c and para-influenza rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and real-time pcr assays for detection of bocavirus in human specimens real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages rapid typing of human adenoviruses by a general pcr combined with restriction endonuclease analysis real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses viral etiology of respiratory infections in children under years old living in tropical rural areas of senegal: the evira project seasonal variation in respiratory syncytial virus infections in children in benin city respiratory syncytial virus infection: denominator-based studies in indonesia association of respiratory picornaviruses with acute bronchiolitis in french infants use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenzalike illnesses in cambodia during three consecutive dry seasons outpatient upper respiratory tract viral infections in children with malaria symptoms in western kenya molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections clinical features of patients with acute respiratory illness and rhinovirus in their bronchoalveolar lavages presence of specific viruses in the middle ear fluids and respiratory secretions of young children with acute otitis media this study was partially funded by a grant from the european society of clinical microbiology and infectious diseases. key: cord- -rwjxk v authors: nan title: asthma & allergy sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: rwjxk v nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- -p pxz a authors: nan title: cystic fibrosis sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: p pxz a nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- - c er a authors: moser, lindsey a.; ramirez-carvajal, lisbeth; puri, vinita; pauszek, steven j.; matthews, krystal; dilley, kari a.; mullan, clancy; mcgraw, jennifer; khayat, michael; beeri, karen; yee, anthony; dugan, vivien; heise, mark t.; frieman, matthew b.; rodriguez, luis l.; bernard, kristen a.; wentworth, david e.; stockwell, timothy b.; shabman, reed s. title: a universal next-generation sequencing protocol to generate noninfectious barcoded cdna libraries from high-containment rna viruses date: - - journal: msystems doi: . /msystems. - sha: doc_id: cord_uid: c er a several biosafety level and/or (bsl- / ) pathogens are high-consequence, single-stranded rna viruses, and their genomes, when introduced into permissive cells, are infectious. moreover, many of these viruses are select agents (sas), and their genomes are also considered sas. for this reason, cdnas and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral rna before transfer out of the bsl- / and/or sa laboratory. this tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (ngs). here, we present a sequence-independent method to rapidly amplify viral genomic rna while simultaneously abolishing both viral and genomic rna infectivity across multiple single-stranded positive-sense rna (ssrna+) virus families. the process generates barcoded dna amplicons that range in length from to , bp, which cannot be used to rescue a virus and are stable to transport at room temperature. our barcoding approach allows for up to barcoded samples to be pooled into a single library and run across various ngs platforms without potential reconstitution of the viral genome. our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular rna, and it can be used to identify pathogens from unknown samples. in summary, we describe a rapid, universal standard operating procedure that generates high-quality ngs libraries free of infectious virus and infectious viral rna. importance this report establishes and validates a standard operating procedure (sop) for select agents (sas) and other biosafety level and/or (bsl- / ) rna viruses to rapidly generate noninfectious, barcoded cdna amenable for next-generation sequencing (ngs). this eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for ngs library construction. ngs data from this sop can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. ing material. our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. keywords: next-generation sequencing, west nile virus, alphavirus, coronavirus, flavivirus, foot-and-mouth disease virus, genomics, picornavirus, rhinovirus s ingle-stranded positive-sense rna (ssrnaϩ) viruses constitute the largest group of viral agents ( ) . even when not encapsidated by viral structural proteins, positivesense rna genomes are sufficient to initiate translation and viral replication upon introduction into permissive cells. the infectious nature of ssrnaϩ viral genomes has made them amenable to reverse genetic manipulation for more than years ( , ) . however, this property is a contributing factor in classifying many ssrnaϩ viruses as select agents (sas) under the federal select agent program ( ) . moreover, many ssrnaϩ viruses are high-priority biosafety level and/or (bsl- / ) pathogens, with a subset listed as potential bioterrorism agents by the u.s. department of health and human services ( , ) . an additional complication is that full-length cdnas from ssrnaϩ viruses that are sas can be used to create rna and rescue/recover the pathogen. thus, in some instances, large cdnas, double-stranded dnas (dsdnas), or clones containing at least two-thirds of the genome may also be regulated as sas. positive-sense rna viruses span multiple virus families, and the infectious nature of these genomic rnas coupled with sa/biosafety/biosecurity concerns inhibit rapid removal from bsl- / containment ( ) or transport and handling of rna samples that are known to contain viral genomes from outbreak settings. this poses a challenge for timely sample processing and sequence analysis, which could significantly hamper responses during outbreak situations. typically, all products generated from infectious material must be proven to no longer contain infectious viral particles or infectious genomic rna prior to transfer to a bsl- space. confirming loss of infectivity (loi) typically occurs via blind infectivity testing, where a subset of the material is placed on a permissive cell line for at least three subsequent passages ( ) . for this reason, transferring cdnas or other nucleic acids from a bsl- / laboratory to a bsl- laboratory is a very difficult and time-consuming process for many laboratories. some investigators have worked out procedures that have been approved by their institutional biosafety committee; however, these procedures vary from institution to institution. for example, some bsl- facilities work with mosquito-transmitted ssrnaϩ viruses including west nile virus (wnv) and chikungunya virus (chikv) that are not considered sas. other bsl- facilities contain ssrnaϩ viruses such as severe acute respiratory syndrome coronavirus (sars-cov) and venezuelan equine encephalitis virus (veev) that are classified as sas, but these facilities have safety protocols that allow transfer of genomic rna or cdna from bsl- to bsl- directly by scientists. finally, there are bsl- facilities working with sas such as foot-and-mouth disease virus (fmdv) at the plum island animal disease center (piadc). here, only trained safety personnel are allowed to transfer cdna samples from high-containment laboratories to bsl- laboratories after inactivation has been carried out under a validated protocol. for each of these scenarios, a robust, universal standard operating procedure (sop) to eliminate infectious material while rapidly generating next-generation sequencing (ngs) libraries is critically needed. fmdv illustrates the majority of the sa, biosafety, and biosecurity issues surrounding high-consequence rna viruses ( ) . among the foreign animal disease viruses, fmdv is the most contagious and has historically set standards for biosafety and biosecurity policies and procedures. the required biosafety level to carry out any infectious virus work with fmdv is biosafety level agriculture (bsl- ag), and currently, the only facility authorized to work with fmdv in the united states is piadc (bsl- ag safety considerations reviewed in references and ) . the fact that the viral rna and rna derived from infected samples can be infectious when transfected or electroporated into susceptible cells results in strict regulation of any nucleic acid derived from fmdvinfected material. currently, the only approved methodology to remove fmdv nucleic acids from the bsl- ag laboratory at piadc involves harsh alkaline and thermal treatment that potentially has a deleterious effect on putative ngs libraries ( ; m. mcintosh, personal communication) . in addition, removal of material derived from diverse bsl- / ssrnaϩ pathogens out of containment requires time-consuming procedures (e.g., multiple blind infectivity passages) to rule out the presence of infectious material. this tremendously limits the capacity to conduct genomic research with viral samples, particularly the application of ngs techniques to understand viral pathogenesis, viral ecology, and vaccine development. for the reasons presented, there is substantial need for a universal sop to generate cdna that rapidly and reproducibly inactivates bsl- / viruses that can be easily assessed by institutional biosafety committees (ibc), which speeds the transfer of nucleic acids from high-containment laboratories to bsl- laboratories, and enables rapid introduction into a variety of ngs pipelines. here, we present a robust sop for generating high-quality, barcoded cdnas directly from genomic rna across multiple virus families. families represented include picornaviridae, alphaviridae, flaviviridae, and coronaviridae, which have genome sizes from~ kb to kb. the strategy builds upon established sequence-independent single-primer amplification (sispa) methods ( ) ( ) ( ) . our data prove that barcoded ngs sequencing libraries can be rapidly generated while simultaneously destroying both viral particle and genomic rna infectivity. our approach is scalable, highly adaptable, and sensitive. the sop generates high-quality sequences spanning the entire genome, up to pooled barcoded samples can be examined in a single ngs run, and the products of the sop work on multiple ngs platforms (e.g., illumina miseq, hiseq, nextseq, and ion torrent). the sop works on starting material of purified virus, tissue culture samples, or tissue samples. we are able to detect virus-specific reads in samples where the input is fewer than pfu and can identify viruses present in an unknown sample. therefore, this application has potential for rapid sequencing of high-titer viral stocks as well as virus discovery and/or forensics. finally, the nonspecific nature of the amplification makes this sop adaptable to negative-strand rna viruses (ssrnaϪ), double-strand rna (dsrna), single-strand dna (ssdna), or double-strand dna (dsdna) viruses. the sop rapidly generates noninfectious, barcoded dna libraries for ssrna؉ viruses. an overview of our approach illustrates nine major steps (fig. a) . first, viral rna is isolated using a commercially available rneasy kit or trizol. second, purified viral rna is used as a template for sequence-independent cdna synthesis, and a random library of barcoded pcr products is constructed. third, dna products of the sop are treated with rnase. fourth, sop products are purified. at this point, we tested all products of the sop for loss of viral infectivity and for the absence of infectious full-length viral genomic rna. this step is not highlighted yellow in fig. and is not included in the final sop, since the methods are virus specific. fifth, immediately prior to transfer, all samples are heated at °c for min to inactivate any potential virus contamination that could inadvertently occur during cdna generation and purification ( ) . sixth, all samples are transferred from bsl- / to bsl- space. seventh, products from the sop are pooled and subjected to ngs library construction. eighth, libraries are sequenced. ninth, sequence data are analyzed and computationally assembled in standard office space. a full detailed protocol for the sop is provided in our supplemental methods (text s in the supplemental material). sequence-independent single-primer amplification (sispa) utilizes a random hexamer primer coupled to a unique barcode (bc-n [ fig. b] ). the bc-n oligonucleotide generates single-stranded cdna from input rna and double-stranded dna by randomly priming the synthesized cdna. finally, a pcr step with primers encoding only the barcode sequence amplifies and uniquely barcodes a sample. as a result, small pcr products are generated with unique barcodes on each terminus of randomly primed amplicons. a representative gel image displays products of the sop obtained from serial dilutions of genomic human rhinovirus (hrv- ) virion rna. at high input rna amounts, a smear between bp and kb is visible. this signal intensity diminishes as the starting material is diluted (fig. c) . upstream steps may be required prior to initiating the sop. when starting from a high-titer virus stock, directly proceeding to the sop (fig. a) will provide the investigator with sufficient sample purity to obtain viral sequence data that spans the majority of the viral genome. for a heterogeneous sample (e.g., virus-infected cells), a host rna depletion step, such as rrna or mrna removal may be required to enrich for virusspecific sequences before proceeding to the sop (fig. d) . the sop recovers full-length genomic sequence data across multiple ss-rna؉ virus families. we sought to perform the sop in multiple laboratories and test diverse virus families. therefore, a standardized version of the sop was distributed to laboratories that have expertise working with various ssrnaϩ virus families ( ) ( ) ( ) . for proof-of-principle experiments, work was performed with both bsl- and bsl- ss-rnaϩ viruses. representative genomic coverage for fmdv, wnv, hrv- , chikv, and middle east respiratory syndrome coronavirus (mers-cov) obtained from illumina miseq sequencing illustrates that . to % genome coverage was achieved for each virus ( fig. a a detailed protocol is provided in text s in the supplemental material. steps in the pink box denote work performed in a biosafety level and/or (bsl- / ) laboratory. steps in the blue box denote work that can be performed in a bsl- laboratory. the asterisk in step indicates that for nonselect agent pathogens (e.g., west nile virus), extracted rna may be moved to bsl- for library construction. step , generating cdna and sispa, utilizes a primer with a random hexamer coupled to a unique barcode (bc-n ). sispa stands for sequence-independent single-primer amplification. (b) the bc-n primer is used for both generating single-stranded cdna from input rna and generating double-stranded dna by randomly priming the synthesized cdna. a pcr step using primers only encoding the barcode sequence with either three or four random nucleotides ( n/ n) at the = end simultaneously amplifies and uniquely identifies (barcodes) a sample. (c) representative gel image that displays products of the sop obtained from serial dilutions of genomic human rhinovirus (hrv- ) virion rna. at high-input rna amounts, a smear between bp and , bp is visible. this signal intensity diminishes as the starting material is diluted. (d) summary of diverse types of starting material which can feed into the sop. samples enriched for virus-specific sequence (e.g., virion stocks) can directly proceed to the sop. for samples that contain a majority of host nucleic acid, the use of upstream procedures to enrich for virus-specific signal (e.g., rrna depletion or mrna enhancement) is recommended. = ends of the genome and large homopolymer regions, such as the poly(c) region present in the = untranslated region ( =-utr) of fmdv ( ) . to date, we have sequenced fmdv samples, mers-cov samples, wnv samples, hrv (both hrv- and - ) samples, and chikv samples (representative data from each family are summarized in table s in the supplemental material). an advantage of this system is the ability to create a single library from hundreds of barcoded samples rather than hundreds of libraries, dramatically reducing the time and cost associated with reagents and labor. in cases where the sequences are of sufficient depth, minor variants that differ from the provided reference sequence may be determined using bioinformatic pipelines that integrate clcbio software ( ) and custom software. representative wnv data ( fig. ; table s ) was subjected to minor variant analysis, and we identified base changes from the reference sequence (genbank accession no. af . ). these changes were a combination of three engineered mutations (positions , , and ) ( ) and eight additional changes present in the virus stock, serving as a control for our sequencing and analyses. we next sought to evaluate the ability of the sop to inactivate infectious genomic rna (grna) and infectious virus. high-titer samples of coronaviruses, flaviviruses, alphaviruses, and picornaviruses were processed, and an aliquot of each sample was used to test for infectious virus or infectious viral grna (see materials and methods and table ). currently, blind passaging of potentially infectious material on permissive cells is a standard assay for infectivity. negative results from blind passages are often required to remove products out of a bsl- facility. this method can detect infectious virus from as little as pfu or from infectious viral grna of limited genomes (table ) . no viral infectivity is present after testing of samples derived from six different ssrnaϩ viruses (table ) . moreover, no genomic rna infectivity is present after testing samples. in all cases, positive-control samples confirmed that each cell line clearly detected both viral infectivity and grna infectivity. while these results suggest that the sop completely inactivates infectious material, an additional heat inactivation step was included to further reduce the risk of residual infectious material leaving bsl- / containment. heat treatment at °c for min completely abolishes infectious hrv- , wnv, and all seven fmdv serotypes (see table s in the supplemental material). heat inactivation for chikv and mers-cov was not performed, but our data indicate the ability of the sop to eliminate infectivity from both enveloped and nonenveloped ssrnaϩ viruses. moreover, high-quality sequence is recovered after heat inactivation of the sispa products for both wnv and hrv- (see fig. s in the supplemental material). it is important to note that for non-sa viruses, the rna may be removed to the bsl- in rna lysis buffer prior to cdna generation if approved by the individual's institutional biosafety committee (ibc). the sop completely abolishes grna infectivity at multiple steps. our initial results demonstrate that the sop removes all grna infectivity from starting material derived from high-titer ssrnaϩ viral stocks. we next initiated experiments to define a specific step in the sop where grna infectivity is abolished, working with the small rna virus hrv- , which is highly infectious when introduced into permissive cells ( ) . we first determined that the limit of detectable rna infectivity for hrv- occurs in the range between . and . pfu on h hela cells (table ). next, grna from hrv- ( ϫ pfu equivalents per sample) were subjected to either the entire sop or stopped at intermediate steps (diagrammed in fig. a ). briefly, replicates for select steps in the sop were transfected into h hela cells. first, purified hrv- rna from pfu was transfected into h hela cells prior to initiating the sop (positive control for transfection). second, the sop was stopped after pcr amplification, and the pcr products were purified. third, the sop was used to generate pcr products, which were treated with l of an rnase cocktail and purified. fourth, the final products from the sop were heat inactivated (which encompasses the entire sop). as a control for the efficacy of the rnase cocktail, rna from pfu of hrv- was treated with the amount of rnase from the sop. material from each group was then transfected into h hela cells and subjected to three blind passages to demonstrate that infectivity was destroyed. results clearly indicate that all infectious grna is removed at the step of pcr (fig. b ), likely resulting from the temperature cycling conditions. however, we cannot positive negative rule out the contribution of rnase h in the cdna synthesis step to the degradation of infectious grna (described in the supplemental sop [text s in the supplemental material]). moreover, the subsequent rnase step and final heat treatment for min at °c serve as additional safety checkpoints. our data suggest that both the pcr step and the downstream rnase cocktail inactivate grna from at least ϫ pfu equivalents for hrv- , proving that the ability of the sop to inactivate grna is extremely robust. to build on the data obtained with hrv- , a similar approach was followed with fmdv ( fig. ) . corroborating the hrv- results, fmdv grna infectivity is abolished post-pcr in the absence of the rnase cocktail (fig. c ). these two experiments highlight that the sop efficiently inactivates all infectious grna at an early step in the sop and that subsequent steps serve as additional fail-safes for inactivation. this is true for both fmdv and hrv- , and supports data in table which clearly demonstrate that the sop abolishes grna infectivity across diverse virus families. to determine the sensitivity of the sop across multiple sequencing platforms, we sequenced identical libraries generated from the sop with both miseq and hiseq. representative data from a serial dilution experiment shows that the limit of detection (lod) for hrv- -specific sequencing reads was approximately . pfu (fig. ) . the hiseq platform produces an average of -to -fold-more sequencing reads for each sample. however, these additional reads do not improve the overall genome coverage nor the limit of detection (fig. ) . therefore, we conclude that the miseq platform is able to sequence all viral reads present in each library and that the hiseq platform simply derives more sequences from the same starting material. evaluating the sensitivity of the sop from purified virions, purified viral genomic rna, and mixed samples. we next sought to further define the sop limits of detection on a miseq platform and directly compare this sensitivity to sequencespecific quantitative real-time reverse transcriptase pcr (qrrt-pcr). separate experiments with dilutions of hrv- virions and hrv- grna in both pure (cell-free supernatant containing virions) and mixed (cellular rna spiked with hrv- ) samples were performed. for test (fig. a) , a virion dilution series precedes rna extraction, followed by the sop. for test (fig. b) , virion rna was extracted from a high-titer stock, followed by serial dilution, and aliquots from each dilution were subjected to the sop. for test (fig. c ), virion dilutions described in test were mixed with hela cells. total rna was extracted from each tube, and rrna was removed before proceeding to the sop. for test (fig. d) , aliquots from each dilution of viral rna described in test were added to g of total hela cell rna. rrna was removed, and the material was subjected to the sop. representative miseq data (black line) from each test is displayed in fig. a to d as the number of hrv- -specific sequencing reads per sample. prior to initiating the sop, an aliquot of diluted rna from each sample was analyzed by qrrt-pcr to determine the input viral rna copy number (red line). this input value provides a comparison for the sequence read output. the approximate qrrt-pcr and miseq lods for this experiment are as follows: qrrt-pcr lod of . however, the sop has the advantage over qrrt-pcr of requiring no a priori knowledge of the sequence of interest and has the benefit of providing sequence information from both known and unknown samples. we further evaluated the ability of the sop to detect wnv in the context of cells or whole tissue. limiting dilutions of wnv-infected cells were spiked into uninfected cells (fig. a ) or uninfected tissues (fig. b ). our method detects wnv-specific reads from as few as infected cells spiked into uninfected cells (fig. a ) and as few as infected cells spiked into uninfected tissues (fig. b) . another experiment was designed to determine the ability of the sop to detect wnv-specific sequencing reads in both acutely and persistently infected mouse tissues. for this study, the sop was performed to generate libraries from the footpad (site of inoculation) and the brain (target organ of wnv) at days , , and ; both tissues support viral production with infectious virus present for at least month after inoculation ( , ) . samples were pooled and sequenced on eight hiseq lanes to ensure sufficient sequencing depth for a proper analysis, yielding between and million reads per sample. viral loads peak in the footpad at day and diminish by day (fig. c) , while peak brain titers occur at day postinoculation (fig. d) . furthermore, virus-specific reads were detected in the brain of one animal at days postinfection, which underscores the power of the sop to detect low levels of virus in a natural infection. the sop can accurately identify "unknown" samples. to test the ability of the sop to recover viral sequence from an unknown sample source, three viral stocks were subjected to the sop, anonymized, and shipped to the j. craig venter institute (jcvi) for sequencing and data analysis in a blind manner (fig. a ). raw data underwent de novo assembly, and large contigs (Ͼ bp) were used to identify the best full-length viral genome references by a nucleotide blast search. sequencing reads were then mapped onto the selected reference genome, and mapping coverage was determined (fig. ) . for st. louis encephalitis virus (slev), western equine encephalomyelitis virus (weev), and chikungunya virus (chikv), we obtained more than % genome coverage, clearly demonstrating the ability of the sop to rapidly identify ssrnaϩ viruses from samples without prior information. in addition, we successfully identified an "unknown" sample, which was a mix of four different viral rnas (wnv, slev, weev, and chikv) (data not shown). the core objective of a biosafety program is the containment of potentially harmful biological and infectious agents. standardization of lab practices at all bsls are defined ( ), but the transfer of potentially infectious material out of high-containment facilities is very challenging and is sometimes the source of containment breaches. likewise, during outbreak situations, the need to handle samples containing potentially infectious viral genomes (or genetic material falling under sa regulations) under high containment may significantly hinder public health responses. in this report, we demonstrate the development of a robust sop for generating high-quality cdnas from ssrnaϩ viruses, which can be used in highly parallel processes to quickly and safely remove samples from bsl- / containment for subsequent genomic sequencing or other similar procedures in bsl- laboratories. protocols to generate ngs sequencing libraries for ssrnaϩ viruses do exist. for example, a recent report ( ) describes a method for fmdv whole-genome sequencing, but it was not proven to destroy infectious material. in addition to complete genome coverage, our data confirm that full-length rnas, cdnas, or dsdnas from these processes cannot be used to recover/ rescue infectious viruses. we demonstrate that this sop completely inactivates viruses from diverse virus families classified as sas or considered high-consequence pathogens. the sop removes infectious genomic rnas and simultaneously amplifies barcoded cdnas that cannot be used to rescue/recover viruses (table and fig. ). the sop also generates high-quality cdna libraries, and the resulting ngs data covers almost entire ssrnaϩ viral genomes from samples tested ( fig. and ) . the sispa method is unable to fully define viral genomic termini, since the method utilizes random hexamer priming. therefore, additional protocols, such as rapid amplification of cdna ends (race) are required for this sequence information. the procedure is sequence independent and can be utilized on both known and unknown viruses (new/emerging), regardless of prior genome information. sequence independence also enables simultaneous identification of multiple pathogens in a sample (e.g., coinfections) or even modified, manufactured pathogens. the described strategy is efficient, scalable, and inexpensive and requires only reagents and equipment that are commonly used in bsl- / laboratories, whereas ngs sequencing equipment is expensive and difficult to maintain, especially within high-containment laboratories. this sop builds on the established sispa method ( , ) to create small overlapping fragments that are barcoded (i.e., uniquely tagged) by the addition of nucleotides to the termini. our barcoding system has many advantages. (i) the barcoded dnas cannot be used to recover/rescue viruses, so they can be safely transferred to bsl- sequencing centers onsite or shipped to large-scale sequencing centers. (ii) our system allows for the pcr amplification of the genomic material (thus, limited amounts of sample or low copy number can yield whole genomes). (iii) it can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for ngs library construction. collaboratively, the sop has been performed more than , times in multiple laboratories to demonstrate that full-length genomic sequences can be obtained, and it can be used to detect very small amounts of viral nucleic acid approaching the sensitivity of qrrt-pcr. the strategy can be applied to diverse starting material such as cell culture supernatants, infected-cell monolayers, and animal tissues. while much of the representative data shown was derived from illumina miseq, the sop can be applied to multiple platforms, including illumina hiseq and ion torrent personal genome machine (pgm). the pgm provides a rapid turnaround time, which allows for viral sequence data to be obtained in hours compared to longer times required for illumina platforms. an interesting result from this study was the observation that both the illumina miseq and hiseq platforms have similar limits of detection on identical ngs libraries (fig. ) . this is likely because the miseq platform sequences most virus-specific molecules in the library, and the hiseq platform only adds more reads from the same starting material. it is important to note that the hiseq platform is still relevant for host gene expression analysis, since additional sequencing depth is critical to provide adequate coverage across all mammalian mrnas. we are also able to directly compare the sensitivity of the sop to sequence-specific quantitative qrrt-pcr methods (fig. ) . while qrrt-pcr is more sensitive, it requires prior knowledge of the sequence of interest, while sispa does not. further, we can detect hrv- -specific sequence from both limiting amounts of hrv- virion rna and from samples where the majority of the sequence data are host derived. we demonstrate the ability to detect low levels of wnv-specific sequence from persistently infected mouse tissues as late as days postinfection (fig. ) . future studies will optimize the sop to improve assay sensitivity to further reduce sequencing cost in samples with limited starting material. however, these data illustrate the value of this protocol for virus discovery and/or forensic analyses. extensive effort was directed toward proving the sop abolishes viral and genomic infectivity across virus families. our experimental design is sufficient to state with high confidence that the sop abolishes infectivity because of the following. (i) the input infectious dose for each sample is high. (ii) the limit of detection for each infectivity test is low. (iii) the sample size is sufficiently high. it is important to note that to assign a confidence interval for this assay, there must be some failures or variability in the final outcome (e.g., cultures positive for infectivity during loss of infectivity testing). our processing of all samples since the initiation of the experiment has a success rate of % (table ) . the ability to demonstrate that all rna infectivity is lost after the pcr step for both hrv- and fmdv (fig. ) strongly suggests that a final rnase step serves as an additional safeguard to remove infectious rna. finally, the heat step of °c for min ensures no residual infectivity due to lab contamination is present prior to transfer from bsl- to bsl- (see table s and fig. s in the supplemental material). however, this step is considered fail-safe, as we demonstrate that infectivity is abolished after the pcr step (fig. ) . applying ngs to sequencing rna viruses represents an unparalleled capacity to generate large amounts of sequence data, which can be used to identify consensus sequences as well as minor sequence variants, or quasispecies, present in a viral population ( ) . limited fmdv population diversity studies explore both intrasample variation during serial passage and the presence of fmdv quasispecies within an infected animal ( , ) ; therefore, the described sop is especially promising for fmdv genomic analyses. to overcome the cost and time limitation of using conventional ngs protocols for fmdv studies in large data sets, a previous study combined ngs and sanger data with sequences available in public databases to study microevolutionary processes of fmdv populations at multiple scales ( ) . a protocol to generate consensus level genome sequences for fmdv and a few other positive-sense polyadenylated rna viruses has been reported ( ) , but it was exclusively tested on an illumina miseq platform. in contrast, our sop provides extra versatility and cost efficiency, as it has been adapted to multiple sequencing platforms, and it allows analysis of up to barcoded samples in a single run. therefore, the scalability and cost-efficiency of the sop described here make it a promising approach to examine quasispecies from large data sets, and it can provide sufficient sequencing depth to examine minor variants from high-titer stocks (see table s in the supplemental material). consistent with previous observations ( ) , we also found lowest coverage in highly structured or repetitive regions of the =-utr. further optimization of the sop is required to investigate low-level viral populations or structurally difficult genomic areas from all starting material ( ) . rapidly evolving ngs technologies have improved our ability to discover novel viral sequences across diverse sample types ( , ) . transcriptomics, analyzed historically through microarray and more recently by ngs, represent another approach to identify both known and novel viral sequences in a given sample (reviewed in reference ). alternative virus discovery approaches have utilized degenerate primers for specific virus families, virus-specific probes ( ) , and random primers for signal amplification and subsequent ngs library construction ( , ) . our sispa approach couples a barcode sequence and random hexamer in a single oligonucleotide and requires no prior knowledge of the sample composition (fig. ) . moreover, hrv- data demonstrate the ability to obtain hrv- -specific reads from grna corresponding to fewer than pfu (fig. ) . importantly, the final products of the sop are small dsdna fragments flanked by sequence barcodes and therefore can be safely and stably shipped to a sequencing facility in the absence of a cold chain. in summary, this universal sop for cdna production that reproducibly inactivates infectivity of bsl- / viruses will speed the transfer of nucleic acids from highcontainment laboratories to bsl- laboratories where ngs can be rapidly performed. this sop is suitable for any pathogen, because it is sequence independent and can be utilized on both known and unknown agents and will simultaneously identify multiple agents. finally, our collection of data suggests that our procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. prior to the generation of sequence data, an sop was established for work at each institution. these institutions include the j. craig venter institute (jcvi), university of wisconsin-madison, plum island animal disease center (piadc), university of maryland school of medicine, and university of north carolina-chapel hill. a detailed, standardized protocol describing all procedures and reagents is supplied in the supplemental material. briefly, total rna was extracted from viral stocks, virus-infected cells, or virus-infected tissues. random hexamer oligonucleotides coupled with unique barcodes (denoted bc-n ) were used for first-strand cdna (life technologies) and second-strand dna (new england biolabs) synthesis. the first-strand cdna synthesis step also includes treatment with rnase h (catalog no. m s; new england biolabs) (see text s in the supplemental material). a second primer, specific for each barcode but lacking a random hexamer, was then used to generate pcr fragments ranging in size from approximately to , bp (life technologies). an example of a bc-n primer sequence is =-tagtacactctagagcactannnnnn- =, and the corresponding pcr primer sequence is =-tagtacactctagagcacta- =. in order to overcome the problems associated with low diversity in the first cycles of illumina reads ( ), we modified the sispa protocol by adding either n's or n's to the = ends of the sispa pcr primers for each barcode, and using an equimolar combination of these modified primers during the pcr amplification step of the protocol. a list of bc-n and pcr primer sequences has been described previously ( ) . all sop products were treated with an rnase cocktail (life technologies) and purified (qiagen). a subset of samples were subjected to heat inactivation at °c for min. all bsl- samples that required the loss of virus and rna infectivity were subjected to testing consistent with the specific institutional biosafety standards. final products of the sop were shipped to jcvi, pooled, and sequenced on the illumina miseq (two -bp paired end [pe] sequencing reads), illumina hiseq (two -bp pe), illumina nextseq (two -bp pe), or ion torrent ( chip -bp kit) platforms. postsequencing genomic analyses. a combination of jcvi-developed and clcbio-developed command line tools were used to process read data from all ngs runs. duplicate reads were removed, reads were demultiplexed based on each sample's specific barcode sequence, and reads were both sequence trimmed to remove the barcode and random hexamer and quality trimmed to remove low-quality bases. sispa barcodes are designed to have a minimum edit distance of between any two barcodes, such that during demultiplexing, two errors may be allowed when searching for barcode sequences at the termini of reads. for barcode demultiplexing, we used bespoke software available from http://sourceforge.net/ projects/deconvolver/ to identify sispa barcode sequences, bin and trim the barcode and random hexamer, but other software packages may be used as well. for samples with a known input sequence, reads were mapped to a prespecified reference sequence. for samples with an unknown input sequence, reads were de novo assembled, and the resulting contigs longer than nucleotides (nt) were used to search for the most appropriate reference sequences available in genbank, using ncbi's blastn against the nt database, with a filter for viruses (taxonomy identifier [id] [taxid] ). once the closest references were chosen, reads were mapped to the selected references. for all analyses, the number of reads that mapped to the references, the percent genome coverage from these mappings, and the number of unmapped reads were determined. when sufficient sequence depth was available, single nucleotide polymorphisms (snps) were determined using jcvi custom software. this pipeline applies statistical tests to minimize false-positive snp calls that could be caused by the types of sequencespecific errors (sse) that may occur in illumina reads previously identified and described ( ) . once a minimum minor allele frequency threshold and significance level are established by the user, the number of minor allele observations and major allele observations in each direction and the minimum minor allele frequency threshold are used to calculate a p value based on the binomial distribution cumulative probability, and if the p values calculated in each of the two sequencing directions are both less than the bonferroni-corrected significance level, then the snp call is accepted. for our analyses, we used a significance level of . (bonferroni corrected for tests in each direction to . ), and a minimum minor allele frequency threshold of %. fmdv methods. (i) fmdv stocks and viral infectivity studies. for fmdv, transcribed rna from fmdv a cruzeiro infectious clone ( ) and lfbk ␣v␤ cells were used to assess the presence or absence of viral infectivity ( ) . lfbk ␣v␤ cells were used to assess the presence or absence of viral infectivity as previously described ( ) . cells were propagated using dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and antibiotics and incubated at °c in % co ( ) . briefly, t flasks seeded with lfbk ␣v␤ cells for h were rinsed and inoculated with sispa products in ml of serum-free medium. after h of adsorption, ml of medium with % serum were added to the flasks, and the flasks were incubated for h at °c on a rocking platform. samples in which no infectivity was observed were amplified through three blind passages. (ii) fmdv rna infectivity studies. bhk- cells (baby hamster kidney cell strain , clone ; atcc ccl ) were used for electroporations. cells were maintained in minimal essential medium (mem) containing % tryptose phosphate broth, % calf serum, and % antibiotics and nonessential amino acids (gibco-brl/invitrogen). sispa products or fmdv rna were electroporated as previously described ( ) . the electroporation products were subsequently passaged on lfbk ␣v␤ cells as described above to assess viral infectivity for a total of three blind passages. to determine the limit of recovery for our system, we electroporated serial dilutions of quantified recombinant wild-type a rna ( ) . the electroporation products were passaged a minimum of three times on lfbk ␣v␤ cells, with infectivity visually checked daily. (iii) rna isol ation, cdna synthesis, and fmdv detection by qpcr. total rna was isolated from supernatants of cell lysates previously electroporated with viral rna, cdna, or sispa using either the qiagen rneasy kit (valencia, ca) or the magmax- viral rna isolation kit (ambion) on a kingfisher magnetic particle processor (thermo scientific) for high-throughput analysis. rna was treated with rnase-free dnase i (sigma-aldrich). superscript iii reverse transcriptase (rt) (life technologies) was used for cdna synthesis following the manufacturer's instructions. cdna was diluted : and run in duplicate following as described previously for fmdv ( ) using path-id quantitative pcr (qpcr) reagents (applied biosystems) in a thermocycler (applied biosystems). samples were considered positive when threshold cycle(c t ) values were Ͻ . (iv) fmdv thermal stability study. a virus stock of fmdv a was prepared at ϫ pfu/ml. quadruplicate tubes were heated at °c for min, °c for min, and °c for min or °c for min (control). samples were then stored at Ϫ °c for further titration by plaque assay ( ) . wnv methods. (i) wnv stocks and rna isolation. two stocks of wnv were used in the studies. one stock (wnv k) was a biological isolate. another stock (fl-wnv) was derived from an infectious cdna clone of wnv k, as previously described ( ) . fl-wnv contains three silent mutations engineered into the infectious clone and, thus, can be differentiated from wnv k ( ) . titers of virus stocks were determined on african green monkey kidney cells (vero; atcc ccl- ). rna was isolated from samples with the qiagen rneasy kit according to the manufacturer's instructions. the initial sample volume was l, and isolated rna was eluted in l rnase-free water. all animal studies that resulted in tissue sample harvest were approved by iacuc at the university of wisconsin-madison. rna was isolated from tissues using a combination of trizol reagent (life technologies) and the qiagen rneasy kit. tissue sections ( mm by mm by mm) from the brains, spleens, and livers of mice were placed in . ml trizol with a . -inch diameter steel ball and homogenized with a qiagen mixer mill for min at cycles/s. the process was repeated as needed until complete homogenization was observed. the samples were incubated for min at room temperature (rt) prior to the addition of l of chloroform. samples were mixed, incubated for min at rt, and centrifuged for , ϫ g for min at °c. the aqueous fraction was transferred to a fresh tube, mixed with l of cold % ethanol, and added to an rneasy column, and rna was extracted following the manufacturer's instructions. rrna was depleted from g of total rna using the ribozero magnetic kit (epicentre, madison, wi) according to the manufacturer's instructions. (ii) wnv viral infectivity studies. rna was isolated as described above, and sispa libraries were generated according to the universal standard operating procedure. one to three sispa libraries were pooled, and l of the pool was added to ml virus diluent (mem plus % fbs). a negative-control sample (diluent alone) and a positive-control sample containing pfu were prepared in parallel. the -ml samples were inoculated into a t flask of vero cells ( ) and incubated at °c and % co for h, rocking every min. five milliliters of complete medium (mem plus % fbs) was added to each flask, and the cells were incubated at °c and % co for days. monolayers were monitored daily for cytopathic effect (cpe). if cpe was apparent, aliquots were collected and frozen at Ϫ °c, and the sample was no longer passaged. if no cpe was present days postinoculation (dpi), ml of supernatant from each flask was inoculated onto fresh vero cells, initiating the infection process again for a total of three passages. to determine the limit of detection for infectious virus, -fold dilutions of wnv ( to Ϫ pfu) were inoculated onto vero cells and subjected to four rounds of blind passaging as described above. the experiment was run in triplicate. cpe was evident in all cells infected with at least pfu by the second passage. mild cpe developed in one flask infected with . pfu at the fourth passage. no cpe was observed in cells infected with less than . pfu. (iii) wnv rna infectivity studies. the presence of infectious wnv rna in sispa libraries was assayed by electroporation of bhk- cells followed by serial passage on vero cells. bhk- cells were trypsinized, washed, and resuspended in pbs at a concentration of . ϫ cells/ml in preparation for electroporation. cells ( . ml) were mixed with l of water, l of sispa pool, or l of water plus l of wnv rna (isolated from virus stock as described above) in a . -mm-gap electroporation cuvette. the cell mixture was pulsed in an electroporator (biorad, richmond, ca) three times at v for . s with -s rests. the cells were incubated for min at rt before they were transferred to a flask containing ml warmed complete medium. the cells were grown at °c and % co and were observed daily for cpe. after days, ml of supernatant from each flask was inoculated onto vero cells, and the samples were serially passaged as described above. to determine the limit of rescue for infectious rna in our system, bhk- cells were mixed with -fold dilutions of wnv rna ( to genomic equivalents [ge]) of wnv and electroporated in triplicate. ge were calculated from isolated viral rna using real-time rt-pcr ( ) . cell supernatants were inoculated onto vero cells and subjected to four blind passages. cpe rapidly developed in cells that were transfected with or ge, while no cpe developed in cells transfected with less than ge. sispa libraries were considered free from infectious virus or rna if no cpe was observed by the end of the third passage on vero cells. as a secondary test for infectious material, l of supernatant from the final passage of each sample was inoculated onto vero cells in duplicate. the infection was allowed to continue for days, at which time monolayers were fixed and examined for virus antigen by immunofluorescence. monolayers were stained with mouse hyperimmune ascitic fluid (cdc; : ) followed by fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse (kpl; : ) and visualized with a nikon eclipse ts fluorescence microscope. viral antigen was observed in all wells inoculated with positive-control wnv samples but was absent in all wells inoculated with negative-control or sispa library supernatant. hrv- and hrv- methods. (i) hrv viral stocks and quantitative pcr. the pwr . molecular clone was obtained from the atcc to rescue hrv- viral stocks, while the pr . plasmid was obtained from the atcc to generate hrv- viral stocks (genbank accession no. l ). supernatants containing infectious virus for both hrv- and hrv- were isolated and aliquoted. the genbank reference sequence for hrv- was used to design a quantitative reverse transcriptase pcr (qrt-pcr) taqman primer/probe set: taqman forward primer, =-ggttaaatggatgttaagaattatatcagctatggttata- =; taqman reverse primer, =-catccaatcagtgttaaagtggcaat- =; taqman probe, -carboxyfluorescein (fam)-cagatccgcaaacaat. to isolate rna, supernatant after each passage was frozen at Ϫ °c and subsequently subjected to rna extraction using a qiagen rneasy kit. rna was treated with rnase-free dnase i. reverse transcription was performed with superscript iii reverse transcriptase (invitrogen). as a negative control to monitor contamination, water was used in place of superscript iii reverse transcriptase, and in each case, reverse transcription was performed with oligo(dt) priming. cdna was diluted to : and run in duplicate using the taqman custom hrv- custom primers and probe on the applied biosystems platform. (ii) hrv rna infectivity studies. h hela cells (crl- ) were used to assess the presence or absence of viral infectivity as visualized by cpe. cells were propagated in six-well plates using dmem supplemented with % fetal bovine serum and incubated at °c in % co for h. rna transfection was performed using l of rna, cdna, or sispa product following the transit-mrna transfection kit protocol (mirus). after transfection, cells were incubated at °c for h, and cpe was observed daily. samples determined to be cpe negative were used in passage . specifically, l of supernatant from passage was used to infect fresh cells at °c for h. after h, . ml of dmem ( ϫ) with % fbs was added to each well, and the cells were incubated at °c for h. cpe was observed daily. samples deemed cpe negative were used in passage following the same protocol as that used for passage . for all experiments, an hrv- genomic rna was transfected in parallel as a positive control for rna infectivity. for large-scale loss of rna infectivity studies, the sop was performed on hrv- samples and hrv- samples in a -well format. briefly, . l from each well was used to make eight tubes of . l (pooling material from a -a , b -b , c -c , etc.). h hela cells were propagated in petri dishes using dmem supplemented with % fetal bovine serum. next, . l of sispa products was transfected into each petri dish using the transit-mrna transfection kit (mirus). cells were incubated at °c for at least h and monitored daily for the appearance of cpe. if no cpe observed, ml of supernatant from each petri dish was inoculated into fresh hela cells. this was repeated for a total of three passages. for all experiments, an hrv- genomic rna was transfected in parallel as a positive control for rna infectivity. to determine the limit of detection for infectious rna, rna was isolated from -fold dilutions of hrv- virion particles. rna was transfected onto h hela cells using the transit-mrna transfection kit (mirus) and subjected to three rounds of blind passaging. cpe was evident in all cells infected with at least . pfu by the second passage. no cpe was observed in cells infected with less than . pfu (data not shown). the limit of infectivity for infectious rna was reproducible over multiple replicates. (iii) hrv viral infectivity studies. for studies of large-scale loss of viral infectivity, the sop was performed on hrv- samples and hrv- samples in a -well format. the final eluted volume was l and was divided into four aliquots. to reduce the amount of cpe testing, samples in all rows were pooled prior to cpe testing. briefly, . l from each well was used to make eight tubes of . l (pooling material from a -a , b -b , c -c , etc.). target h hela cells were propagated in t flasks using dmem supplemented with % fetal bovine serum and incubated at °c. for infectivity studies, the supernatant was removed, and the pooled . l was mixed with ml of infection medium ( ϫ dmem plus % fbs). this mix was used to infect t flasks for h, gently rocking flasks every min. after h, the medium was replaced, and flasks were incubated at °c for h. if no cpe was observed, ml of supernatant from each flask was inoculated into fresh hela cells. this was repeated for a total of three passages. for all experiments, infectious hrv- viral particles served as a positive control for cpe. to determine the limit of detection for infectious virus, -fold dilutions of hrv- were inoculated onto h hela cells and subjected to three rounds of blind passaging as described above. cpe was evident in all cells infected with at least . pfu by the second passage. no cpe was observed in cells infected with less than . pfu (data not shown). the limit of infectivity for infectious virus was reproducible over multiple replicates. alphavirus methods (alphavirus stocks, sispa reactions, and infectivity assays). alphavirus sispa testing was done using either an infectious-clone-derived girdwoods.a. strain of sindbis virus (sinv) ( ) or an infectious-clone-derived vaccine strain ( / ) of chikv ( ) , which was generously provided by terry dermody, vanderbilt university. these infectious clones were engineered to express the mkate fluorescent protein followed by the fmdv a protease in frame between the viral capsid and e gene as previously described ( ) . all virus stocks were generated by production of virus length genomic rna using sp mmessage mmachine rna transcription kits (ambion), which was electroporated into bhk- cells as described previously ( ) . titers of each viral stock were determined using our standard plaque assay protocol on vero cells ( ) . sispa reactions were tested for the presence of infectious sinv or chikv on vero cells. briefly, l of the pooled sispa library was mixed with ml of complete mem (mem plus % fbs, l-glutamine [l-glut], and penicillin-streptomycin [pen-strep]), and the vero cells were placed in t flasks for h, with rocking every min at °c and % co . at the end of the -h incubation, ml of complete medium was added to the flask. a mock control flask received complete medium alone. the first-passage monolayers (p: ) were maintained for h, with the monolayers assessed for cytopathic effect and red fluorescence daily. at h, % of supernatant from the passage flask was introduced onto a new monolayer (p: ) using the same -h infection protocol, and the flasks were maintained for h, with the monolayers assessed for cpe and red fluorescence every h. next, % of the p: supernatant was then placed on a third monolayer (p: ) and monitored for an additional h. to assess assay sensitivity, the cpe protocol was also performed using ml of complete medium containing , , , or . pfu of sinv or chikv. the assay sensitivity routinely ranged from . to pfu. to test alphavirus rna inactivation in sispa reactions, bhk cells were electroporated with l of the pooled sispa sinv or chikv libraries, and seeded into t flasks with ml mem supplemented with % fbs, l-glut, and pen-strep. monolayers were maintained for h at °c and assessed for cpe and red fluorescence every h. at h, ml ( %) of supernatant was transferred to a new t flask of vero cells (p: ), which were monitored for cpe every day for h, at which point % of the supernatant from p: was placed on a third monolayer (p: ) of vero cells, which was assessed for cpe for h. vero e (monkey kidney epithelial cells, atcc crl- ) for plaque assays were grown in minimal essential medium (corning) supplemented with % (vol/vol) fbs (sigma aldrich), % l-glutamine (gibco) and % penicillin-streptomycin (gemini bio-products) at °c in a % co atmosphere. (ii) mers-cov quantification and safety testing. to determine the titers of live mers-cov, media from infected cells and processed rna were placed on vero e cells to determine the titer in pfu per milliliter as previously described ( ) . to quantify the infectivity of samples throughout the described rna isolation protocol, rna or cdna intermediates or final purified products were used to infect vero e cells in a six-well plate ( ϫ cells per well). the cells were passaged every h for blind passages for each well, and cpe was examined. supplemental material for this article may be found at http://dx.doi.org/ . / msystems. - . figure s , eps file, mb. classification and nomenclature of viruses. fifth report of the international committee on taxonomy of viruses cloned poliovirus complementary dna is infectious in mammalian cells rna virus reverse genetics and vaccine design guidance on the regulation of select agent and toxin nucleic acids. division of select agents and toxins host factors in positive-strand rna virus genome replication neurological disorders, virus persistence and hypomyelination in calves due to intra-uterine infections with bovine virus diarrhoea virus us department of health and human services early events in the pathogenesis of foot-and-mouth disease in cattle after controlled aerosol exposure foot and mouth disease. oie world organisation for animal health facility design considerations for select agent animal research select agent and toxin regulations: beyond the eighth edition of the guide for the care and use of laboratory animals inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat viral genome sequencing by random priming methods metagenomic analysis of rna viruses in a fresh water lake sispa-seq for rapid whole genome surveys of bacterial isolates thermal inactivation of foot-and-mouth disease viruses in suspension dendritic cell immunoreceptor regulates chikungunya virus pathogenesis in mice persistence of west nile virus in the central nervous system and periphery of mice repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection a universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated rna viruses using the illumina miseq deep sequencing identifies noncanonical editing of ebola and marburg virus rnas in infected cells infectious cdna clone of the epidemic west nile virus from new york city role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates next generation sequencing of viral rna genomes beyond the consensus: dissecting withinhost viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing evolution of foot-and-mouth disease virus intrasample sequence diversity during serial transmission in bovine hosts observing micro-evolutionary processes of viral populations at multiple scales ocean plankton. structure and function of the global ocean microbiome combining metagenomics, metatranscriptomics and viromics to explore novel microbial interactions: towards a systems-level understanding of human microbiome application of next-generation sequencing technologies in virology virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis large scale loss of data in low-diversity illumina sequencing libraries can be recovered by deferred cluster calling molecular characterization of a foot-and-mouth disease virus containing a -nucleotide insertion in the =untranslated region a continuous bovine kidney cell line constitutively expressing bovine alphavbeta integrin has increased susceptibility to foot-and-mouth disease virus genetically engineered foot-and-mouth disease viruses with poly(c) tracts of two nucleotides are virulent in mice analysis of a foot-andmouth disease virus type a isolate containing an sgd receptor recognition site in vitro and its pathogenesis in cattle use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus foot-and-mouth disease virion rna: studies on the relation between the length of its =-poly(a) segment and infectivity virus detection protocols for west nile virus in vertebrate and mosquito specimens identification of adult mouse neurovirulence determinants of the sindbis virus strain ar reovirus cell entry requires functional microtubules stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease a mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence growth and quantification of mers-cov infection we thank sarah carter for critical reading of the manuscript and tad kochel for technical input.this project has been funded under department of homeland security contract hshqdc- -c-b . this project has been funded under department of homeland security contract hshqdc- -c-b . key: cord- - pu x rj authors: etemadi, mohammad reza; jalilian, farid azizi; othman, norlijah; lye, munn-sann; ansari, sara; yubbu, putri; sekawi, zamberi title: diversity of respiratory viruses detected among hospitalized children with acute lower respiratory tract infections at hospital serdang, malaysia date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: pu x rj background: the role of respiratory viruses as the major cause of acute lower respiratory tract infections (alrtis) in children is becoming increasingly evident due to the use of sensitive molecular detection methods. the aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with alrtis. methods: the cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (rsv), human metapneumovirus (hmpv), influenza virus a and b (ifv-a and b), parainfluenzavirus , , and (piv , , and ), human rhinoviruses (hrv), human enterovirus (hev), human coronaviruses (hcov) e and oc , human bocavirus (hbov) and human adenovirus (hadv) in hospitalized children with alrtis, at hospital serdang, malaysia, from june to december , . the study was also designed in part to assess the performance of the conventional methods against molecular methods. results: viral pathogens were detected in ( . %) of the patients. single virus infections were detected in ( . %) patients; ( . %) were co-infected with different viruses including double-virus infections in ( . %) and triple-virus infections in ( . %) cases. approximately % of samples were found to be positive using conventional methods compared with % using molecular methods. a wide range of respiratory viruses were detected in the study. there was a high prevalence of rsv ( . %) infections, particularly group b viruses. other etiological agents including hadv, hmpv, ifv-a, piv – , hbov, hcov-oc and hev were detected in . , . , . , . , . , . and . percent of the samples, respectively. conclusion: our results demonstrated the increased sensitivity of molecular detection methods compared with conventional methods for the diagnosis of artis in hospitalized children. this is the first report of hmpv infections in malaysia. background: the role of respiratory viruses as the major cause of acute lower respiratory tract infections (alrtis) in children is becoming increasingly evident due to the use of sensitive molecular detection methods. the aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with alrtis. methods: the cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (rsv), human metapneumovirus (hmpv), influenza virus a and b (ifv-a and b), parainfluenzavirus , , and (piv , , and ), human rhinoviruses (hrv), human enterovirus (hev), human coronaviruses (hcov) e and oc , human bocavirus (hbov) and human adenovirus (hadv) in hospitalized children with alrtis, at hospital serdang, malaysia, from june to december , . the study was also designed in part to assess the performance of the conventional methods against molecular methods. results: viral pathogens were detected in ( . %) of the patients. single virus infections were detected in ( . %) patients; ( . %) were co-infected with different viruses including double-virus infections in ( . %) and triple-virus infections in ( . %) cases. approximately % of samples were found to be positive using conventional methods compared with % using molecular methods. a wide range of respiratory viruses were detected in the study. there was a high prevalence of rsv ( . %) infections, particularly group b viruses. other etiological agents including hadv, hmpv, ifv-a, piv - , hbov, hcov-oc and hev were detected in . , . , . , . , . , . and . percent of the samples, respectively. conclusion: our results demonstrated the increased sensitivity of molecular detection methods compared with conventional methods for the diagnosis of artis in hospitalized children. this is the first report of hmpv infections in malaysia. most respiratory tract infections are caused by viruses or bacteria with viruses causing the highest proportion of infections. the major causes of acute respiratory infections (ari) in children are respiratory syncytial virus (rsv), parainfluenzavirus (piv), influenza virus (ifv), adenovirus (adv), and human rhinoviruses (hrv). human metapneumovirus (hmpv), human coronaviruses (hcov) hku , nl , e and hospitalized children is cost-effective. it is pivotal in directing active treatment early in the course of the illness following detection, reducing unnecessary antibiotic prescription, and limiting nosocomial transmission to high-risk patients (adcock et al., ; lanata et al., ; osiowy, ; woo et al., ) . in addition, assessment of the morbidity of specific etiological agents of alrtis identified using sensitive detection methods in hospitalized patients is important to determine agent-specific interventions such as vaccination against rsv (lanata et al., ) . extensive detection of infections will also expand our knowledge of the etiology of pneumonia and assist in determining which etiological agents should be considered for vaccine development (rudan et al., ) . recent developments in molecular diagnosis of respiratory viruses and the discovery of new viruses have renewed the interest in respiratory virus epidemiology. however, there is still a considerable deficiency in the diagnosis of viruses which cause alrti (ieven, ) . far too little attention has been paid to the epidemiology of respiratory viral infections in malaysia. therefore, in this study, the goal was to detect a panel of classical and newly discovered respiratory viruses including ifv, rsv, piv, adv, hmpv, hrv, hev, hcov, and hbov which cause alrti in children below years of age at hospital serdang. both conventional methods including direct immunofluorescence assay, cell culture and shell vial culture and molecular diagnostic techniques including multiplex pcr and sequencing were used. the survey was conducted at two -bed pediatric wards in hospital serdang, a government-funded multi-specialty hospital located in the district of sepang in the state of selangor, malaysia. the participants were children more than one-month-old and less than years of age who were admitted to the hospital between june , and december , with the diagnosis of alrti. patients with congenital or acquired immunosuppressive conditions, with conditions that posed a potential hazard in obtaining the nasopharyngeal samples (e.g. bleeding diathesis, severe respiratory compromise) as determined by the clinicians and children with incomplete data or inadequate samples were excluded from the study. all potential subjects (including careers of the patients) were briefed on the study before written informed consent was obtained by the pediatrician in-charge of the case. approval from the following authorities was obtained prior to the start of the study: the ministry of heath malaysia research and ethics committee (mrec) (nmrr- - - ), the medical research ethics committee, faculty of medicine and health sciences, upm (upm/fpsk/pads/t -mjketikaper/f (jmpp fep ( ) )) and the research ethics board of hospital serdang were obtained. blood samples for bacterial culture were collected by the nurses involved in the study and sent to the department of pathology, hospital serdang. nasopharyngeal aspirate (npa) was taken through both nostrils by inserting a disposable catheter (no. or no. ) connected to a mucus extractor. npas were transported in viral transport medium (vtm) to the laboratory and refrigerated at °c- °c until required. in order to avoid repeated freezing and thawing, all npas were processed upon receipt. the samples were vortexed vigorously for s and centrifuged at ×g for min. the supernatant was collected and set aside for virus isolation and genome extraction. the cell pellet was used in a direct immunofluorescence assay (dfa) after washing several times to remove mucus to avoid nonspecific fluorescence. most of the samples were detected with dfa at the same day of samples received followed by cell culture and genome extraction and cdna synthesis. the pcr reaction was performed when a bunch of samples were available. detection of the viruses was performed concurrently during the six months of the study period. the d ultra direct immunofluorescence assay respiratory virus screening & identification kit (diagnostic hybrids inc. (dhi), usa) which contains a blend of murine fluorescein isothiocyanate (fitc)conjugated monoclonal antibodies (mabs) was used as the first step to detect eight common viruses including rsv, hmpv, ifv type a and b, piv - , hadv. dfa negative samples were inoculated onto shell vial culture (svc). r-mix tm ready cells (dhi, usa), ready-to-use mixed cell monolayers comprising mink lung cells (mv lu) and human adenocarcinoma cells (a ), were used according to the manufacturer's recommendations. viral rna/dna was extracted from the filtered supernatant of npa using magmax viral rna isolation kit (applied biosystems, ambion, usa) according to the manufacturer's instructions. the concentration and purity of the extracts (a /a nm and a /a nm) were measured using a nanodrop (thermofisher scientific, usa). the first strand cdna synthesis was carried out on rna extracts in a final volume of μl by random hexamer primer using revertaid ™ h minus first strand cdna synthesis kit (fermentas, usa) following the manufacturer's instructions. the samples were incubated first for min at °c followed by min at °c. the reaction was terminated by heating at °c for min. three multiplex rt-pcr (mp/rt-pcr - ) and subsequently two hemi-nested multiplex pcr assays (hnmp/pcr - ) were carried out for the molecular detection of rna viruses (bellau-pujol et al., ) . mp/rt-pcr targeted influenza viruses a and b, rsv (types a and b), hmpv (a and b). mp/rt-pcr detected parainfluenza virus types , , and (a and b) (piv - ). the mp/rt-pcr contained primers for the detection of hrv, hev, hcov oc and e. an internal control consisting of glyceraldehyde- -phosphate dehydrogenase (gapdh) was included in mp/rt-pcr . hbov and hadv were detected in the samples by singleplex pcr and nested pcr respectively (allander et al., a; lu and erdman, ) . primer set hadvhexf /adhexr and nested primer set adhexf /adhexr were used to detect hadv hexon gene hyper-variable regions - (hvr − ). the pcr products were separated by electrophoresis in a . % agarose gel and visualized using ethidium bromide under uv light. the multiplex pcr was validated using pcr ® . -topo ® plasmid vector [topo ta cloning® kit (invitrogen, usa)]. hep- (atcc ccl- , usa), mrc- (atcc ccl- , usa), vero (atcc (ccl- , usa) and hela cells (atcc, usa) were purchased from american type culture collection (atcc) and cultured according to their guidelines. all rsv positive samples were cultured on vero and hep- cells. hadv positive samples were inoculated onto hela and hep- cells. mrc- and hela cells were used for samples which were positive for hrv. cell cultures with characteristic cpe for rsv and hadv were harvested and confirmatory testing was performed with dfa. the tube cultures showing cpe for hrv were harvested and confirmed by pcr. a second blind passage was performed after a week for cultures without characteristic cpe. data were analyzed using spss version . . all p-values were twotailed and p-values of < . were considered statistically significant. comparisons between the results obtained by molecular methods and conventional methods were evaluated by mcnemar's test and pairedsamples t-test. a total of children less than five years of age who fulfilled the inclusion criteria as outlined above and were hospitalized with alrtis during a -week period between june , and december , were enrolled in the study. dfa was the first conventional method of detection of common respiratory viruses used to identify eight viruses including ifv a & b, pivs - , rsv, hmpv and hadv. hrv, hev, hcovs, piv and hbov were excluded from immunological detection because of a lack of specific monoclonal antibodies. for each virus the pattern of immunofluorescent staining was specific and was used for confirmatory purposes. eighty eight ( . %) of the npa samples were dfa positive as follows: ( . %) for rsv, ( . %) for hmpv, ( . %) for hadvs, ( . %) for ifv a and ( . %) for pivs - . detection of rsv, hmpv, piv - , ifv-a & b, hadv in shell vial culture was attempted as the second stage of the conventional method on the remaining dfa negative npa samples. seven additional viruses were detected and a total of ( . %) samples were virus positive by dfa followed by shell-vial culture. therefore, this method was able to detect some viruses which were missed by dfa: rsv and one case each for hadv, hmpv and piv . for rsv, cpe was detected in of specimens representing a % recovery. twenty-four samples positive for hadvs were inoculated into hela and hep- cell lines. five of samples ( %) showed characteristic cpe. semi-confluent monolayers of hela and mrc- cell lines were used to isolate of the hrv positive samples detected using rt-pcr. the ability of the multiplex method to specifically detect multiple viruses in the same reaction tube was evaluated by testing a mixture of cloned plasmids of targeted viruses. analysis of the pcr products showed that each multiplex method simultaneously detected all three control viruses included in each reaction as well as the internal control with the expected band sizes. in the presence of all primer sets in the multiplex reaction, no mispriming was observed in the positive and negative control tubes. the specificity of the mp/rt-pcr products was confirmed by nucleotide sequence analysis. three multiplex rt-pcr (mp/rt-pcr - ) and subsequently hemi-nested multiplex pcr (hnmp/pcr - ) were carried out on nucleic acids extracted from clinical specimens. the specific products were clearly separated and identified on a . % seakem agarose gel, both for virus control and for clinical specimens. in total, samples ( . %) from the panel of were virus positive and ( . %) specimens were virus negative using this method. almost all viruses ( %) were detected in the first stage of the mp/rt-pcr - assay. in the second stage six ( / , %) additional viruses were detected by hnmp/pcr - . using normal and nested pcr, of the samples tested, were found to be positive for hbov and ( . %) for adenovirus. of these positive samples, single infections were documented in one hbov and three cases of hadv infection. in total, samples ( . %) were positive for respiratory viruses using the molecular method while ( . %) were negative. gapdh was successfully amplified from all of the npa samples tested by pcr indicating that there were no pcr inhibitors in the reactions. therefore, false negative results were excluded using this internal control. one hundred and twelve patients ( . %) were found to be infected with a single virus with the most frequently detected viruses being rsv ( %), hrv ( %), ifv-a ( . %) and hmpv ( . %)(table ). no single infection due to hcovs was observed. multiple respiratory viral infections were documented in ( %) samples; consisting of ( . %) double infections and ( . %) triple infections. the most frequently detected viruses in these patients were rsv ( , . %), followed by hadv ( , . %) and hrv ( , . %). dual infections of rsv with hadv and hrv were the most prevalent multiple viral infections found in the study ( / , % and / , %, respectively). culture of blood samples from patients revealed bacterial infections in only five ( %) cases including one patient with a single infection ( . %) with ἀ-hemolytic streptococcus viridans. four other samples were also virus positive and were considered to be nosocomial infections (blood cultures results were positive after h) as follows: rsv/burkholderia cepacia, hrv/ b. cepacia and hrv/ coagulase-negative staphylococcus. m. pneumonia infections were not identified in any of the patients. in total, specimens ( . %) from the panel of were virus positive by a combination of conventional and molecular methods, and seven ( . %) specimens were virus negative. the comparison between conventional and molecular methods is depicted in table . a greater number of samples ( . %) were found to be virus positive using molecular methods compared to conventional methods ( . %) (p < . , mcnamara's test). there were ( . %), ( . %) and ( . %) more positive samples compared with dfa, dfa plus svc and dfa plus svc plus conventional cell culture methods, respectively. on the other hand molecular assays were able to detect more viruses (p < . , paired-samples t-test). the monthly distribution of cases with respiratory tract viruses is shown in fig. . during the study period, a continuously persisting activity was seen for rsv, hrv, hadv, and hmpv. influenza a was detected from july to september, with a peak in august followed by a plateau from september onwards. an increased incidence of hrv and rsv cases was seen after the influenza. a peak from september onwards, peaking in october. for the viruses with low incidence, no distinct pattern was seen. recent developments in molecular diagnostics and the discovery of new viruses have created a renewed interest in the epidemiology of (van den hoogen et al., ) . regional determination of the epidemiology of specific viral infections will improve the treatment guidelines for doctors (irmen and kelleher, ) . the main goal of the current study was to detect a broad panel of respiratory viruses associated with hospitalized children with alrtis in malaysia. we report a high prevalence of respiratory viruses in hospitalized children (allander et al., ; jartti et al., ; jennings et al., ; richard et al., ) . this was achieved using highly sensitive nested pcr which was applied to a broad spectrum of viruses. npa samples from ( . %) patients were positive for single and/or multiple viruses. single virus infections were detected in approximately two-thirds of the samples compared with almost one-third of samples which were found to contain multiple viruses. the performance of conventional diagnostic methods and molecular methods was also evaluated using the samples in this study. as reported in other studies (coiras et al., (coiras et al., , freymuth et al., ; lasala et al., ; weinberg et al., ) we established that molecular methods were more sensitive than conventional methods for the detection of respiratory viruses in children hospitalized with alrtis. many respiratory viruses including rsv-a, and b, ifv-a, piv , piv , piv , hmpv, hrv-a and c, hev, hadv, hbov, and hcov-oc were detected in the patients in this study. rsv was the most prevalent virus detected in % of samples. previous publications have frequently identified rsv as the major viral pathogen associated with lrti in children (chan et al., ; grimwood et al., ; hall et al., ; richard et al., ; zamberi et al., ) . our results provide further supporting evidence that rsv infection is a frequent cause of hospitalization among children in tropical and developing countries (weber et al., ; who, ) . hrv was the second most prevalent virus and was detected in one-third of patients which is a similar infection rate to that reported in other studies (chung et al., ; kim and hodinka, ; lau et al., ; linsuwanon et al., ; miller et al., miller et al., , papadopoulos et al., ; peltola et al., ) . rsv and hrv have also been reported as the most common causes of lrtis in other studies (calvo et al., ; franz et al., ; gruteke et al., ; jennings et al., ; papadopoulos et al., ) . the finding of the current study is also consistent with study by nathan who found rv and hrv as the most detected virus associated with alrtis in a prospective study in malaysia (nathan et al., ) . co-infections with other viruses were found in approximately % ( / ) of hrv infections. the high prevalence of hrv in this study suggests that virus testing should be routinely aq hadv was the third ( . %) most common virus detected in this study. adenoviruses are responsible for . - % of all lrtis occurring in infants and children (chen et al., ; jennings et al., ; john et al., ; lee et al., ; rocholl et al., ) . our findings seem to be consistent with the detection rate of % among hospitalized children with alrtis in argentina (videla et al., ) . this high detection rate is especially important in developing countries with high prevalence of measles and malnutrition. hmpv was detected as the fourth most prevalent virus with an infection rate of %. this is consistent with a . - % detection rate among otherwise healthy children hospitalized with lrtis in several other studies (foulongne et al., a; lee et al., ; williams et al., ; wolf et al., ) . we demonstrated for the first time that hmpv could be an important cause of lrti in children in malaysia. the positivity rate is also comparable with a recent study of children hospitalized with alrti in subtropical brazil ( . %) (oliveira et al., ). the prevalence of hbov was between . % (bastien et al., ) to % (allander et al., b in these children with respiratory infections. in our study, hbov was detected for the first time in malaysia in of nasopharyngel aspirates giving a prevalence of . % (etemadi et al., ) . the detection rate was comparable to that ( . %) reported in france (foulongne et al., b) and sweden ( . %) (allander et al., b) but was lower than that reported ( . %) in singapore (tan et al., ) . sensitive multiplex pcr assays give useful information about the presence of multiple pathogens and their epidemiological and clinical effects (bellau-pujol et al., ) . in the current study, all of the multiple infections were diagnosed exclusively using pcr, which further supports the superiority of molecular methods over conventional methods (rovida et al., ; van de pol et al., ) . evaluation of the relative importance of each coexisting agent may play an important role in understanding the etiopathogenesis of these viruses (tsolia et al., ) . identification of all infectious agents is especially important in high-risk immonocompromised patients for appropriate antiviral therapy (leland and ginocchio, ) . in this study, multiple viral infections were found in % of patients and were usually combinations of rsv with hadv and/or hrv. the co-detection rate is similar to that found by calvo ( ) (calvo et al., ) continued presence of all of the surveyed viruses during the study period with no distinct seasonality. rsv and hadv was the most commonly detected co-infection. the high proportion of hadv co-infections ( . %) with other viruses in this study is comparable with ( %) hadv co-infection rates in a study by calvo ( ) (calvo et al., ) . the second most prevalent combination was rsv and hrv ( %). this combination has been reported as the most common coinfection in other studies (chung et al., ; papadopoulos et al., ; paranhos-baccalà et al., ; richard et al., ) . the high incidence of rsv and hrv co-infection can be explained by the substantial overlapping of the monthly distribution observed for these viruses during the study period (paranhos-baccalà et al., ) . quantification of the viruses in the samples may help to better understand the etiological role of each virus (rovida et al., ) . a study of the clinical features is required to clarify the disease severity in mixed infections (paranhos-baccalà et al., ) . a wide range of respiratory viruses were detected in this study facilitated by molecular diagnostic methods. molecular methods increased the detection rate by up to % compared with conventional methods. the high detection rate of hrv confirmed its association with severe lrti and hospitalization. our study also demonstrated that hmpv and hbov can be important causes of hospitalization of malaysian children. yearly variations in the incidence of respiratory viruses may influence their association with alrtis and therefore our six months study should be considered as a snapshot of viral alrtis in pediatric inpatients. effect of rapid viral diagnosis on the management of children hospitalized with lower respiratory tract infection cloning of a human parvovirus by molecular screening of respiratory tract samples cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus infection development of three multiplex rt-pcr assays for the detection of respiratory rna viruses detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region viral aetiology of lower respiratory tract infection in young malaysian children respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in - detection of viruses identified recently in children with acute wheezing simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested pcr assays first detected human bocavirus in a malaysian child with pneumonia and pre-existing asthma: a case report human metapneumovirus infection in young children hospitalized with respiratory tract disease human bocavirus in children correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness risk factors for respiratory syncytial virus bronchiolitis hospital admission in new zealand practical implementation of a multiplex pcr for acute respiratory tract infections in children the burden of respiratory syncytial virus infection in young children currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections use of monoclonal antibodies for rapid diagnosis of respiratory viruses in a community hospital respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections etiology of acute respiratory infections in children in tropical southern india respiratory virus infections serious respiratory illness associated with rhinovirus infection in a pediatric population methodological and quality issues in epidemiological studies of acute lower respiratory infections in children in developing countries prospective comparison of r-mix (tm) shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants clinical severity of respiratory adenoviral infection by serotypes in korean children over consecutive years ( - ) role of cell culture for virus detection in the age of technology high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease molecular typing of human adenoviruses by pcr and sequencing of a partial region of the hexon gene detection of respiratory viruses by molecular methods rhinovirus-associated hospitalizations in young children human rhinovirus c associated with wheezing in hospitalised children in the middle east viruses and hospitalization for childhood lower respiratory tract infection in malaysia: a prospective study epidemiology and genetic variability of human metapneumovirus during a -year-long study in southeastern brazil direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-pcr assay association of rhinovirus infection with increased disease severity in acute bronchiolitis mixed respiratory virus infections rhinovirus infections in children: a retrospective and prospective hospital-based study the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis adenoviral infections in children: the impact of rapid diagnosis monoclonal antibodies versus reverse transcription pcr for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital epidemiology and etiology of childhood pneumonia human rhinovirus group c in hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection genomic analysis of adenovirus isolated from argentinian children with acute lower respiratory infections virological investigations in sudden unexpected deaths in infancy (sudi) superiority of reverse transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children acute respiratory infections: influenza (update human metapneumovirus infection in children hospitalized for wheezing comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients respiratory viruses detected in hospitalised paediatric patients with respiratory infections this work was partially supported by grants from the ministry of science, technology and innovation malaysia (grant number ). we are grateful to all of the doctors and nurses of the pediatric department, hospital serdang for providing nasopharyngeal aspirates for the study. the authors declare that they have no competing interests. key: cord- -fxvvndic authors: liu, n.; chee, m. l.; koh, z. x.; leow, s. l.; ho, a. f. w.; guo, d.; ong, m. e. h. title: machine learning dimensionality reduction for heart rate n-variability (hrnv) based risk stratification of chest pain patients in the emergency department date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: fxvvndic background: chest pain is among the most common presenting complaints in the emergency department (ed). swift and accurate risk stratification of chest pain patients in the ed may improve patient outcomes and reduce unnecessary costs. traditional logistic regression with stepwise variable selection has been used to build risk prediction models for ed chest pain patients. in this study, we aimed to investigate if machine learning dimensionality reduction methods can achieve superior performance than the stepwise approach in deriving risk stratification models. methods: a retrospective analysis was conducted on the data of patients > years old who presented to the ed of singapore general hospital with chest pain between september and july . variables used included demographics, medical history, laboratory findings, heart rate variability (hrv), and hrnv parameters calculated from five to six-minute electrocardiograms (ecgs). the primary outcome was -day major adverse cardiac events (mace), which included death, acute myocardial infarction, and revascularization. candidate variables identified using univariable analysis were then used to generate the stepwise logistic regression model and eight machine learning dimensionality reduction prediction models. a separate set of models was derived by excluding troponin. receiver operating characteristic (roc) and calibration analysis was used to compare model performance. results: patients were included in the analysis, of which ( %) met the primary outcome of -day mace. patients with mace were older and more likely to be male. all eight dimensionality reduction methods marginally but non-significantly outperformed stepwise variable selection; the multidimensional scaling algorithm performed the best with an area under the curve (auc) of . . all hrnv-based models generated in this study outperformed several existing clinical scores in roc analysis. conclusions: hrnv-based models using stepwise logistic regression performed better than existing chest pain scores for predicting mace, with only marginal improvements using machine learning dimensionality reduction. moreover, traditional stepwise approach benefits from model transparency and interpretability; in comparison, machine learning dimensionality reduction models are black boxes, making them difficult to explain in clinical practice. stepwise variable selection; the multidimensional scaling algorithm performed the best with an area under the curve (auc) of . . all hrnv-based models generated in this study outperformed several existing clinical scores in roc analysis. conclusions: hrnv-based models using stepwise logistic regression performed better than existing chest pain scores for predicting mace, with only marginal improvements using machine learning dimensionality reduction. moreover, traditional stepwise approach benefits from model transparency and interpretability; in comparison, machine learning dimensionality reduction models are black boxes, making them difficult to explain in clinical practice. keywords: machine learning; dimensionality reduction; heart rate n-variability (hrnv); heart rate variability (hrv); chest pain; emergency department background chest pain is among the most common chief complaints presenting to the emergency department (ed) [ ] [ ] [ ] . the assessment of chest pain patients poses a diagnostic challenge in balancing risk and cost. inadvertent discharge of acs patients is associated with higher mortality rates while inappropriate admission of patients with more benign conditions increases health service costs [ , ] . hence, the challenge lies in recognizing low-risk chest pain patients for safe and early discharge from the ed. there has been increasing focus on the development of risk stratification scores. initially, risk scores such as the thrombolysis in myocardial infarction (timi) score [ , ] and the global registry of acute coronary events (grace) score [ ] were developed from post-acs patients to estimate short-term mortality and recurrence of myocardial infarction. the history, electrocardiogram (ecg), age, risk factors, and initial troponin (heart) score was subsequently designed for ed chest pain patients [ ] , which demonstrated superior performance in many comparison studies on the identification of low-risk chest pain patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nonetheless, the heart score has its disadvantages. many potential factors can affect its diagnostic and prognostic accuracy, such as variation in patient populations, provider determination of low-risk heart score criteria, specific troponin reagent used, all of which contribute to clinical heterogeneity [ ] [ ] [ ] [ ] . in addition, most risk scores still require variables that may not be available during the initial presentation of the patient to the ed such as troponin. there remains a need for a more efficient risk stratification tool. we had previously developed a heart rate variability (hrv) prediction model using readily available variables at the ed, in an attempt to reduce both diagnostic time and subjective components [ ] . hrv characterizes beat-to-beat variation using time, frequency domain and nonlinear analysis [ ] , and has proven to be a good predictor of major adverse cardiac events (mace) [ , , ] . most hrv-based scores were reported to be superior to timi and grace scores while achieving comparable performance with heart score [ , , , ] . recently, we established a new representation of beat-to-beat variation in ecgs, the heart rate n-variability (hrnv) [ ] . hrnv utilises variation in sampling rr-intervals and overlapping rr-intervals to derive additional parameters from a single strip of ecg reading. as an extension to hrv, hrnv potentially supplements additional information about adverse cardiac events while reducing unwanted noise caused by abnormal heart beats. moreover, hrv is a special case of hrnv when n = . the hrnv prediction model, developed from multivariable stepwise logistic regression, outperformed the heart, timi and grace scores in predicting -day mace [ ] . nevertheless, multicollinearity is a common problem in logistic regression models where supposedly independent predictor variables are correlated. they tend to overestimate the variance of regression parameters and hinder the determination of the exact effect of each parameter, which could potentially result in inaccurate identification of significant predictors [ , ] . in the paper, hrnv parameters were derived but only seven variables were left in the final prediction model, and this implies the possible elimination of relevant information [ ] . within the general medical literature, machine learning dimensionality reduction methods are uncommon and limited to a few specific areas, such as bioinformatics studies on genetics [ , ] and diagnostic radiological imaging [ , ] . despite this, dimensionality reduction in hrv has been investigated and shown to effectively compress multidimensional hrv data for assessment of cardiac autonomic neuropathy [ ] . in this paper, we attempted to investigate several machine learning dimensionality reduction algorithms as possible alternatives to stepwise selection, hypothesizing that these algorithms could be useful in preserving useful information while improving prediction performance. we aimed to compare the performance of hrnv-based dimensionality reduction models against hrnv stepwise logistic regression model and conventional risk stratification tools such as the heart, timi, and grace scores, in the prediction of -day mace in chest pain patients presenting to the ed. a retrospective analysis was conducted on data collected from patients > years old who presented to singapore general hospital ed with chest pain between september to july . these patients were triaged using the patient acuity category scale (pacs) and those with pacs or were included in the study. patients were excluded if they were lost to the -day follow-up or if they presented with st-elevation myocardial infarction (stemi) or non-cardiac aetiology chest pain such as pneumothorax, pneumonia and trauma as diagnosed by the ed physician. patients with ecg findings that precluded quality hrnv analysis such as artefacts, ectopic beats, paced or non-sinus rhythm were also excluded. for each patient, hrv and hrnv parameters were calculated using hrnv-calc software suite [ , ] from a five to six-minute single-lead (lead ii) ecg performed via the x-series monitor (zoll medical, corporation, chelmsford, ma). table shows the full list of hrv and hrnv parameters used in this study. in addition, the first -lead ecgs taken during patients' presentation to the ed was interpreted by two independent clinical reviewers and any pathological st changes, t wave inversions and q-waves were noted. patients' demographics, medical history, first set of vital signs and troponin-t values were obtained from the hospital's electronic health records (ehr). in this study, high-sensitivity troponin-t was selected as the cardiac biomarker and an abnormal value was defined as > . ng/ml. the primary outcome measured was any mace within days, including acute myocardial infarction (stemi/non-stemi), emergent revascularization procedures such as percutaneous coronary intervention (pci) or coronary artery bypass graft (cabg), or death. the primary outcome was captured through retrospective review of patients' ehr. dimensionality reduction in machine leaning and data mining [ ] refers to the process of transforming high-dimensional data into lower dimensions such that less features are selected . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint or extracted while preserving essential information of the original data. two types of dimensionality reduction approaches are available, namely feature selection (commonly called variable selection in biostatistics) and feature extraction. feature selection methods generally reduce data dimensionality by choosing a subset of variables, while feature extraction methods transform the original feature space into lower-dimensional space through linear or nonlinear feature projection. in clinical predictive modelling, feature selection techniques such as stepwise logistic regression are popular for constructing prediction models [ ] . in contrast, feature extraction approaches [ ] are less commonly used in medical research, although they have been widely used in computational biology [ ] , image analysis [ , ] , physiological signal analysis [ ], among others. in this study, we investigated the implementation of eight feature extraction algorithms and evaluated their contributions to prediction performance in risk stratification of ed chest pain patients. we also compared them with a prediction model that was built using conventional stepwise variable selection [ ] . henceforth, we use the terms "dimensionality reduction" and "feature extraction" interchangeably. given that there were samples ( , ), = , , … , , in the dataset ( , ), where each sample had original features/variables and its label = or , with indicating a positive primary outcome, i.e. mace within days. we applied dimensionality reduction algorithms to project into a -dimensional space ( < ). as a result, the original dataset ∈ ℝ × became ̂∈ ℝ × . there was a total of = candidate variables in this study. as suggested in liu et al. [ ] , some variables were less statistically significant in terms of contributions to the prediction performance. thus, we conducted univariable analysis and preselected a subset of ̃ variables if their <̃, where ̃ was a threshold determined by predictive performance using pca [ ], the most common technique for linear dimensionality reduction. in this study, we implemented eight dimensionality reduction algorithms, including pca, kernel pca ( in this study, we chose logistic regression as the classification algorithm to predict the mace outcome. before applying all eight dimensionality reduction algorithms, we ran pca and logistic regression through -fold cross-validation to determine the threshold ̃ to preselect a subset of ̃ variables, such that the prediction performance in terms of area under the curve (auc) was the largest. we did this preselection to ensure the removal of less significant variables as shown by univariable analysis, after which ∈ ℝ × became ̃∈ ℝ ×̃. in summary, the inputs to all dimensionality reduction algorithms were in ̃dimensional space. subsequently, conventional logistic regression was implemented to take -dimensional ̂ to predict , where -fold cross-validation was used. to compare machine learning dimensionality reduction and traditional stepwise variable selection, we implemented our previously built logistic regression model [ ] , in which the following variables were used: age, diastolic blood pressure, pain score, st-elevation, stdepression, q wave, cardiac history (the "history" component in the heart score), . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint troponin, hrv nn , hr v skewness, hr v sampen, hr v apen, hr v apen, hr v rmssd, hr v skewness, and hr v hf power. the predictive performances of eight models built from different dimensionality reduction algorithms and the model obtained using convention stepwise variable selection [ ] were compared with the receiver operating characteristic (roc) curve analysis, and the corresponding sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) measures were reported. the calibration plots of all nine prediction models were also generated. all analyses were conducted in python version . . (python software foundation, delaware, usa). we included chest pain patients in this study, of which ( %) patients had mace within days of presentation to the ed. table presents the baseline characteristics of the patient cohort. patients with mace were older (median age years vs. years, p = . ) and more likely to be male ( . % vs. . %, p = . ). history of diabetes, current smoking status, and pathological ecg changes such as st elevation, st depression, t wave inversion, pathological q waves, and qtc prolongation were significantly more prevalent in patients with the primary outcome. troponin-t and creatine kinase-mb levels were also significantly elevated in patients with the primary outcome. there was no statistically significant difference in patient ethnicity between mace and non-mace groups. figure (a) depicts the pca-based predictive performance versus the threshold ̃ (for preselection of variables) and figure (b) shows the number of preselected variables versus threshold ̃. the predictive performance peaked at ̃= . , where a total of variables were preselected, including gender, diastolic blood pressure, pain score, st-elevation, stdepression, t-wave inversion, q wave, cardiac history, ekg and risk factor components of the heart score, troponin, hrv rmssd, hrv nn , hrv pnn , hrv hf power, hrv poincaré sd , hr v rmssd, hr v nn , hr v pnn , hr v hf power, hr v poincaré sd , hr v rmssd, hr v nn , hr v hf power, hr v poincaré sd , hr v rmssd, hr v hf power, hr v poincaré sd , hr v rmssd, and hr v poincaré sd . these were used as inputs to all dimensionality reduction algorithms whose outputs were linear or nonlinear combinations of these variables. shows the roc curves of the eight dimensionality reduction algorithms, the stepwise logistic regression, and three clinical scores. all eight dimensionality reduction methods marginally outperformed the stepwise variable selection, and mds performed the best with an auc of . . table presents roc analysis results of all methods/scores where sensitivity, specificity, ppv, and npv are reported with % confidence intervals (cis). figure presents the calibration curves of predictions by all methods/scores. the stepwise model and seven dimensionality reduction models (pca, kpca, lsa, grp, srp, mds, and isomap) showed reasonable model calibrations, in which their curves fluctuated . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint along the diagonal line, meaning these models only slightly overestimated or underestimated the predicted probability of -day mace. the lle model was unable to achieve good calibration. in comparison, all three clinical scores (heart, timi, and grace) generally underpredicted the probability of -day mace. figure shows roc curves of hrnv models without using cardiac troponin. at feature dimensions of , , , , , , , and , the highest auc values of pca, kpca, lsa, grp, srp, mds, isomap, and lle were . , . , . , . , . , . , . , and . , respectively. the stepwise model without troponin yielded an auc of . compared to . with troponin. all hrnv-based prediction models outperformed both the timi and grace scores, while achieving marginally better results than the heart score. in this study, we showed that machine learning dimensionality reduction yielded only marginal, non-significant improvements compared to stepwise variable selection in predicting the risk of -day mace among chest pain patients in the ed. this corroborates with similar observations that traditional statistical methods can perform comparably to machine learning algorithms [ , ] . among the models integrated with troponin, the mds model had the best performance (auc of . , % ci . - . ), which slightly outperformed the stepwise model (auc of . , % ci . - . ). among the models that did not include troponin, pca, kpca, lsa, grp, and mds performed equally well, achieving an auc of . , compared with the stepwise model without troponin which had an auc of . . in general, traditional stepwise approach was proved to be equally well as machine learning dimensionality reduction methods in risk prediction, while benefiting from model simplicity, transparency, and interpretability that are desired in real-world clinical practice. high-dimensional data suffers from the curse of dimensionality, which refers to the exponentially increasing sparsity of data and sample size required to estimate a function to a given accuracy as dimensionality increases [ ] . dimensionality reduction has successfully mitigated the curse of dimensionality in the analysis of high-dimensional data in various domains such as computational biology and bioinformatics [ , ]. however, clinical predictive modelling typically considers relatively few features, limiting the effects of the curse of dimensionality. this may account for the relatively limited benefit of dimensionality reduction in our analysis. additionally, despite marginal improvements in performance compared to stepwise variable selection, transparency and interpretability of machine learning dimensionality reductionbased methods are limited as they rely on complex algorithmic transformations of variables to a lower-dimensional space, leading to obstacles in the adoption of such models in realworld clinical settings. in contrast, traditional biostatistical approaches like logistic regression with stepwise variable selection deliver a simple and transparent model, in which the absolute and relative importance of each variable can be easily interpreted and explained from the odds ratio. marginal improvements in performance should be weighed against these limitations in interpretability, which is an important consideration in clinical predictive modelling. comparing the eight dimensionality reduction algorithms, pca and lsa use common linear algebra techniques to learn to create principal components in a compressed data space, while . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint mds, isomap, and lle are nonlinear, manifold learning-based dimensionality reduction methods. as observed from our results, complex nonlinear algorithms did not show obvious advantage over simple pca and lsa methods in enhancing the predictive performance. yet, nonlinear algorithms are more computationally complex and require more computing memory. for example, kpca and isomap have computational complexity of ( ) and memory complexity of ( ), while pca has computational complexity of (̃ ) and memory complexity of (̃ ) [ ] . in applications of clinical predictive modelling, -the number of patientsis usually larger than ̃ -the number of variables; in our study, is and ̃ is or , depending on the inclusion of troponin. this suggests that linear algorithms may be preferred due to reduced computational complexity and memory while retaining comparable performance. another observation in this study was that the impact of preselection (as shown in figure ) on predictive performance was more substantial than that of dimensionality reduction, indicating the importance of choosing statistically significant candidate variables. our study also reiterates the value of hrnv-based prediction models for chest pain risk stratification. among chest pain risk stratification tools in the ed, clinical scores like heart, timi, and grace are currently the most widely adopted and validated [ , ]. however, a common barrier to quick risk prediction using these traditional clinical scores is the requirement of cardiac troponin, which can take hours to obtain. to address these difficulties, machine learning-based predictive models that integrate hrv measures and clinical parameters have been proposed [ , , , ] , including our development of hrnv, a novel alternative measure to hrv that has shown promising results in predicting -day mace [ ] , which was the stepwise model in this paper. both the dimensionality reduction-based predictive models and the stepwise model with troponin presented superior performance than heart, timi, and grace scores. when troponin was not used, several dimensionality reduction-based models such as pca, kpca, and mds still yielded marginally better performance than the original heart score, while benefiting from generating the predictive scores in merely five to six minutes. additionally, table shows that all hrnv-based predictive models had higher specificities than the heart score while all hrnv-based models except isomap also improved on the already high sensitivity of the heart score [ , ]. the specificities of kpca, isomap, and mds were significantly higher by an absolute value of almost percent. substantial improvements to the specificity of mace predictive models may reduce unnecessary admission and thus minimise costs and resource usage [ ] . this is particularly relevant in low-resource settings, for example, the overburdened eds in the current coronavirus disease (covid- ) pandemic, where novel methods in resource allocation and risk stratification could alleviate the strain on healthcare resources[ ]. there remains a need for further investigation into methods that utilise information from the full set of hrv and hrnv variables. from variables in the initial data set, dimensionality reduction performed the best with a preselection of variables, of which were hrv and hrnv parameters. that is, majority of the newly constructed hrnv parameters were removed based on the strict significance threshold of p< . on univariable analysis. therefore, novel hrnv measures were not fully used in prediction models of day mace, leaving room for further investigation of alternative ways of using them. moving forward, it may be valuable to develop and evaluate deep learning frameworks [ ] to synthesize novel low-dimensional representations of multidimensional information. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint we acknowledge the following limitations of this study. first, the clinical application (i.e. risk stratification of ed chest pain patients) was only one example of clinical predictive modelling, thus our conclusion on the effectiveness of machine learning dimensionality reduction algorithms may not be generalizable to other applications, particularly those with a larger number of variables. secondly, only eight most used dimensionality reduction algorithms were investigated, while many other methods are available. last, we did not build a workable predictive model for risk stratification of ed chest pain patients, although the hrnv-based models showed promising results compared with existing clinical scores. we aim to further investigate deep representations of the input variables (both hrnv parameters and clinical information) to achieve more rapid prediction and significantly improve predictive performance. to our knowledge, this was the first study to compare machine learning dimensionality reduction with conventional stepwise variable selection for risk stratification of chest pain patients in the ed. we found that hrnv-based models using stepwise logistic regression performed better than existing chest pain scores for predicting mace, with only marginal improvements using machine learning dimensionality reduction. this demonstrated the clinical applicability of the traditional stepwise prediction model due to its simplicity, transparency, and interpretability. to fully utilise the information (i.e. variables) in building high-performing predictive models, we suggest that additional investigations into methods to better represent the large amount of hrnv variables should be conducted. furthermore, explainable artificial intelligence deserves in-depth exploration such that the derived models are interpretable by clinicians. acs, acute coronary syndrome ami, acute myocardial infarction auc, area under the curve apen, approximate entropy ci, confidence intervals cabg, coronary artery bypass graft covid- , coronavirus disease dfa, detrended fluctuation analysis ecg, electrocardiogram ehr, electronic health records ed, emergency department grp, gaussian random projection grace, global registry of acute coronary events hrnv, heart rate n-variability hrv, heart rate variability hf, high frequency heart, history, ecg, age, risk factors, and initial troponin iqr, interquartile range kpca, kernel principal component analysis lsa, latent semantic analysis lle, locally linear embedding lf, low frequency mace, major adverse cardiac events . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint mean nn, average of r-r intervals mds, multidimensional scaling npv, negative predictive value nn , the number of times that the absolute difference between successive r-r intervals exceeds ms nn n, the number of times that the absolute difference between successive rrni/rrnim sequences exceeds ×n ms pacs, patient acuity category scale pci, percutaneous coronary intervention pnn , nn divided by the total number of r-r intervals pnn n, nn n divided by the total number of rrni/rrnim sequences ppv, positive predictive value pca, principal component analysis roc, receiver operating characteristic rmssd, square root of the mean squared differences between r-r intervals rri, r-r interval sampen, sample entropy sd, standard deviation sdnn, standard deviation of r-r intervals srp, sparse random projection stemi, st-elevation myocardial infarction timi, thrombolysis in myocardial infarction vlf, very low frequency the ethical approval was obtained from the centralized institutional review board (cirb, ref: / /c) of singhealth, in which patient consent was waived. not applicable. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. nl and meho hold patents related to using heart rate variability and artificial intelligence for medical monitoring. nl, zxk, dg, and meho are currently advisers to tiim sg. the other authors report no conflicts. this work was supported by the duke-nus signature research programme funded by the ministry of health, singapore. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. authors' contributions nl conceived the study and supervised the project. nl and mlc performed the analyses and drafted the manuscript. nl, mlc, zxk, sll, afwh, dg, and meho made substantial . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . byrne c, toarta c, backus b, holt t: the heart score in predicting major adverse cardiac events in patients presenting to the emergency department with possible acute coronary syndrome: protocol for a systematic review and metaanalysis. systematic reviews , ( ). . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint table : comparison of performance of the hrnv models (based on -fold cross-validation), heart, timi, and grace scores in predicting -day major adverse cardiac events (mace). the cut-off values were defined as the points nearest to the upper-left corner on the roc curves. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : roc curves (based on the optimal number of dimensions) generated by the stepwise model, eight dimensionality reduction models, and three clinical scores, where the heart rate n-variability based prediction models were built without using cardiac troponin. . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint best clinical practice: current controversies in evaluation of low-risk chest pain-part best clinical practice: current controversies in the evaluation of low-risk chest pain with risk stratification aids. part evaluating chest pain in the emergency department: searching for the optimal gatekeeper missed diagnoses of acute cardiac ischemia in the emergency department state-of-the-art evaluation of emergency department patients presenting with potential acute coronary syndromes the timi risk score for unstable angina/non-st elevation mi: a method for prognostication and therapeutic decision making e: timi risk score for st-elevation prognostic values of risk scores in chinese patients with chest pain: prospective -centre cohort study short-and long-term prognostic utility of the heart score in patients evaluated in the emergency department for possible acute coronary syndrome. critical pathways in cardiology comparing heart, timi, and grace scores for prediction of -day major adverse cardiac events in high acuity chest pain patients in the emergency department comparison of the heart and timi risk scores for suspected acute coronary syndrome in the emergency department comparison of the grace, heart and timi score to predict major adverse cardiac events in chest pain patients at the emergency department integrating heart rate variability, vital signs, electrocardiogram, and troponin to triage chest pain patients in the ed understanding factors that influence the use of risk scoring instruments in the management of patients with unstable angina or non-st-elevation myocardial infarction in the netherlands: a qualitative study of health care practitioners' perceptions. bmc health services research documentation of heart score discordance between emergency physician and cardiologist evaluations of ed patients with chest pain secondary analysis of frequency, circumstances and consequences of calculation errors of the heart (history, ecg, age, risk factors and troponin) score at the emergency departments of nine hospitals in the netherlands heart rate variability risk score for prediction of acute cardiac complications in ed patients with chest pain heart rate variability: a review risk scoring for prediction of acute cardiac complications from imbalanced clinical data an intelligent scoring system and its application to cardiac arrest prediction ) . . % ( . % - . %) . % ( . % - . %) . % ( . % - . %) auc, area under the curve; ci, confidence interval; ppv, positive predictive value; npv, negative predictive value; heart, history, ecg, age, risk factors and troponin; timi global registry of acute coronary events we would like to thank and acknowledge the contributions of doctors, nurses, and clinical research coordinators from the department of emergency medicine, singapore general hospital. table : list of traditional heart rate variability (hrv) and novel heart rate n-variability (hrnv) parameters used in this study. mean nn, average of r-r intervals; sdnn, standard deviation of r-r intervals; rmssd, square root of the mean squared differences between r-r intervals; nn , the number of times that the absolute difference between successive r-r intervals exceeds ms; pnn , nn divided by the total number of r-r intervals; nn n, the number of times that the absolute difference between successive rrni/rrnim sequences exceeds ×n ms; pnn n, nn n divided by the total number of rrni/rrnim sequences; vlf, very low frequency; lf, low frequency; hf, high frequency; sd, standard deviation; sampen, sample entropy; apen, approximate entropy; dfa, detrended fluctuation analysis. key: cord- -kheek lx authors: carroll, kecia n.; gebretsadik, tebeb; minton, patricia; woodward, kimberly; liu, zhouwen; miller, e. kathryn; williams, john v.; dupont, william d.; hartert, tina v. title: influence of maternal asthma on the cause and severity of infant acute respiratory tract infections date: - - journal: j allergy clin immunol doi: . /j.jaci. . . sha: doc_id: cord_uid: kheek lx background: respiratory syncytial virus (rsv) and rhinovirus infections are the most common significant infant respiratory tract illnesses and are associated with increased but differential risks of childhood asthma. objective: we sought to determine whether maternal asthma is associated with higher odds of infant respiratory tract infection with rhinovirus versus rsv and increased infection severity. methods: mother-infant dyads were enrolled from - during an infant respiratory tract infection ( with rhinovirus and with rsv). mothers were classified into mutually exclusive groups (atopic asthma, nonatopic asthma, and no asthma). we determined viral cause using pcr and the severity of the infant’s respiratory tract infection using the bronchiolitis severity score. adjusted relative odds of maternal asthma with viral cause were calculated by using logistic regression. proportional odds models assessed the association of maternal asthma and infant infection severity. results: infants with a mother with atopic asthma compared with infants whose mothers did not have asthma were more likely to have rhinovirus versus rsv infection (adjusted odds ratio, . ; % ci, . - . ). similarly, among infants with rhinovirus, having a mother with atopic asthma was associated with increased infection severity (adjusted odds ratio, . ; % ci, . - . ). this relationship was not seen among infants with rsv. conclusions: clinically significant rhinovirus infection during infancy was more strongly associated with having a mother with atopic asthma than clinically significant rsv infection. having a mother with atopic asthma was associated with increased severity of infant rhinovirus but not rsv infections. infants with rhinovirus were more likely to have a familial atopic predisposition, which might partly explain the subsequent increased asthma risk. bronchiolitis, a lower respiratory tract infection (lrti) commonly caused by respiratory syncytial virus (rsv) and less commonly by human rhinovirus (hrv), affects an estimated % to % of children in the first year of life and is a leading cause of hospitalization during infancy. [ ] [ ] [ ] [ ] in addition to the acute morbidity seen with bronchiolitis, infants hospitalized with bronchiolitis and young children who experience virus-induced wheezing illnesses are at increased risk of recurrent wheezing and asthma in early childhood. [ ] [ ] [ ] [ ] [ ] the pathogenesis of the increased wheezing after viral bronchiolitis is not fully understood. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in efforts to learn whether children at risk of bronchiolitis are also at increased risk for asthma, studies have investigated whether a family history of asthma is associated with the severity or incidence of bronchiolitis during infancy, with some prior studies finding an association, whereas others did not. , , [ ] [ ] [ ] [ ] prospective birth cohorts in which all children have a familial predisposition to asthma, such as a cohort based in perth, australia, and the childhood origins of asthma (coast) cohort, have investigated the association of a viral cause of infections in early life and subsequent wheezing and asthma. , , in the coast cohort hrvinduced wheezing illnesses in early life were found to be stronger predictors of wheezing and asthma at age years than rsvinduced illnesses. , , however, it is not known whether infants with symptomatic hrv infections that lead to an unscheduled health care visit are more likely to have a familial predisposition to asthma than infants with symptomatic rsv infection. in this investigation that included mother-infant dyads enrolled in the tennessee children's respiratory initiative (tcri), we tested the hypothesis that a familial atopic predisposition was associated with viral cause and increased severity of viral acute respiratory tract infection (ari) during infancy. the tcri is a prospective cohort of term (> _ weeks), non-low-birth-weight (> _ g) infants and their mothers designed to investigate the association of characteristics of infant viral aris, such as severity and cause, and familial atopic predisposition on the development of early childhood asthma and atopy. this investigation included the mother-infant dyads in which the infant presented for an unscheduled clinic or emergency department visit or hospitalization and was determined to have a sole hrv-or rsv-induced ari. mother-infant dyads were enrolled at the time of an infant ari during viral respiratory seasons, september through may - . dyads were recruited in the inpatient, emergency department, and clinic settings at a single academic institution, and the children are currently being followed longitudinally through age years to ascertain asthma and atopy outcomes. each woman provided written informed consent for participation of herself and her infant. the vanderbilt university institutional review board approved the study. during study enrollment, research nurses administered an in-person structured questionnaire that included questions regarding demographics, the infant's home environment, the index infant's illness, previous medical history of the infant, and detailed family asthma and atopic disease history, including maternal responses to the international study of asthma and allergies in children questionnaire. at enrollment, research nurses obtained nasal and throat swabs from the infants for viral detection. through a structured medical chart review, information was abstracted regarding the infant's medical visit, including birth weight, room air pulse oximetry, requirement for supplemental oxygen, history of prior wheezing, and detailed medical information. final discharge diagnoses were obtained through chart review after discharge. self-reported maternal asthma status was defined as a positive response to the question ''have you ever had asthma?,'' which was asked as part of the international study of asthma and allergies in children questionnaire and/or to the question ''were you diagnosed with asthma as a child?'' , maternal atopy was determined based on skin prick test results or allergen-specific ige levels. preferentially, women underwent skin prick tests to saline, histamine, and aeroallergens: cat pelt, alternaria species, grass mix # , ragweed mix, oak mix, tricophyton species, mite mix, and cockroach mix (quintest extract tray; hollister-stier, spokane, wash). allergen-specific ige measurement (phadiatop; phadia, kalamazoo, mich) was performed on maternal blood samples for women who could not undergo skin prick tests or who had an inadequate skin test. multiallergen screens for specific ige (phadiatop) were measured with the immunocap (performed by the johns hopkins daci laboratory). a positive phadiatop result was defined as . ku a /l or greater by using the standards in place at the time the assays were performed. , among women for whom allergen sensitization status was determined based on skin prick test results or allergen-specific ige levels, we classified women by whether they reported a history of asthma into mutually exclusive categories: atopic asthma, nonatopic asthma, and no asthma. women with selfreported asthma and evidence of allergen sensitization (> _ positive skin prick test response or positive phadiatop result) were classified as having atopic asthma, women with self-reported asthma and without evidence of allergen sensitization were classified as having nonatopic asthma, and women who did not report self-reported asthma were in the no asthma group. nasal and throat swabs were obtained from infants at the time of enrollment, and the biospecimens were processed, placed in aliquots, and stored at c. the specimens were tested in batches for rsva and b, hrv, adenovirus, human metapneumovirus, coronaviruses, influenza a and b, and parainfluenza types , , and by using real-time rt-pcr with the cepheid smart cycler ii, as previously described. pcr results were used to identify infants with a sole rsv-or hrv-induced ari. infants included in this study had an hrv-or rsv-induced ari, either viral upper respiratory tract infections (urtis) or lrtis. children with a urti had a health care provider's diagnosis of a viral urti and/or symptoms, including fever, cough, congestion, hoarse cry, otitis media, and/or rhinorrhea without evidence of lower respiratory tract symptoms or respiratory distress. infants with a physician's diagnosis of bronchiolitis or wheezing, signs and symptoms consistent with bronchiolitis on chart review, or both were considered to have a viral lrti. , we determined the severity of the ari by using an ordinal bronchiolitis severity score with factors including respiratory rate, room air oxygen saturation, and the presence and extent of wheezing and flaring and retractions. scores range from to , with higher scores indicating more severe illness. , covariates other variables of interest obtained from the questionnaire administered at enrollment included self-reported maternal race/ethnicity, maternal education, secondhand smoke (shs) exposure, infant's insurance type (tennessee medicaid, private, or none), infant's birth weight (in grams), infant's sex, infant's age at enrollment (in weeks), and siblings. descriptions of demographics and characteristics of the infants with sole hrv or rsv infections are presented as frequencies and proportions for categorical variables and medians and interquartile ranges for continuous variables. univariate analyses were conducted to compare maternal asthma factors by the infant's hrv-or rsv-induced ari status by using the wilcoxon rank sum test for continuous variables or the pearson x test for categorical variables. in our analyses we first defined maternal asthma using self-reported asthma in the women. in addition, to examine the association of maternal asthma in combination with an objective measure of atopy, we repeated all analyses using a more detailed definition that classified women as having atopic asthma, nonatopic asthma, or no asthma by incorporating their skin prick test or allergen-specific ige findings. therefore we investigated whether ( ) having a mother with self-reported asthma and ( ) having a mother with atopic or nonatopic asthma was associated with an infant's ari with hrv or rsv. we assessed the association of measures of maternal asthma and virus type in the overall ari group (combined urti and lrti) and next among the lrti subgroup. we applied a logistic regression model with variable hrv( ) or rsv( ) as a binary outcome variable and maternal asthma as defined above, as our main factor along with covariates. because of our limited regression power determined by the hrv( ) group, we used propensity score adjustment to prevent overfitting because the propensity score analysis adjusts for many confounding factors simultaneously while preserving analytical power. variables in the propensity score model included a priori selected variables: maternal race/ethnicity, shs exposure, infant's insurance type, infant's birth weight, infant's sex, infant's age at enrollment, and number of siblings. in our next set of analyses we examined whether having a mother with asthma (first defined by maternal self-report and then using the atopic asthma and nonatopic asthma classifications) was associated with increased severity of the infant's hrv-or rsv-induced ari. we conducted a separate analysis for infants with sole hrv-or rsv-induced ari and in the subgroups with lrtis. we used the proportional odds model to evaluate the association of maternal asthma with infant hrvor rsv severity using the bronchiolitis severity score. for infants with rsv, a priori selected variables in the multivariable j allergy clin immunol volume , number models included maternal race/ethnicity, shs exposure, infant's insurance type, infant's birth weight, infant's sex, infant's age at enrollment, and number of siblings. analyses among hrv-induced lrtis were limited by small sample size for a full covariates model, and therefore we performed a propensity score-adjusted model that included infant's sex, age, and birth weight. we performed an interaction analysis to assess whether the association between maternal asthma and severity of the infant's ari was different depending on the infant's hrv or rsv status. the proportional odds model was used with a cross-product term of maternal history of asthma and virus positivity (rsv and hrv ) and adjustment for covariates. statistical analyses were performed with r version . . software. a total of infants with sole infection with either hrv or rsv were included in this study. table i highlights the demographics and characteristics of the cohort by infant ari cause, as determined by means of pcr: positive for hrv only (n ) or rsv only (n ). compared with infants with rsv, infants with hrv were more likely to be older ( vs weeks, p < . ), have medicaid insurance ( % vs %, p . ), have mothers who were african american ( % vs %, p < . ), have a urti versus an lrti ( % vs %, p < . ), and have a lower median bronchiolitis severity score ( vs . , p < . ). among infants with aris and the lrti subgroup, we determined the association of self-reported maternal asthma and infant hrv-or rsv-induced aris (both urtis and lrtis). infants with hrv were more likely to have a mother with self-reported asthma than infants with rsv ( % vs %, p . , table i ). in adjusted analyses, compared with infants whose mothers did not have asthma, infants with a mother with self-reported asthma had an increased relative odds of having hrv-induced than rsv-induced ari (adjusted odds ratio, . ; % ci, . - . ). in analyses limited to infants with lrtis, infants with hrv were more likely to have a mother with self-reported asthma than infants with rsv ( % vs %, p . ). in adjusted analyses infants with a mother with self-reported asthma had an increased relative odds of having hrv-induced than rsv-induced lrti (adjusted odds ratio, . ; % ci, . - . ). maternal allergen sensitization determined based on skin prick test results or allergen-specific ige levels was available for % of the mothers (n ) and was used to identify maternal atopic asthma. a larger percentage of infants with hrv-induced aris had a mother with atopic asthma than infants with rsv-induced aris ( % vs %, respectively), whereas the percentages of infants with a mother with nonatopic asthma were similar ( % vs %, respectively). having a mother with atopic asthma was associated with increased odds of an infant having hrv versus rsv infection when compared with having a mother without asthma (propensity score-adjusted odds ratio, . ; % ci, . - . ). having a mother with nonatopic asthma compared with no asthma was not associated with viral cause (adjusted odds ratio, . ; % ci, . - . ). a larger percentage of infants with hrv-induced lrtis had a mother with atopic asthma than infants with rsv-induced lrtis ( / [ %] vs / [ %]); however, the percentages of infants with a mother with nonatopic asthma were similar ( / [ %] vs / [ %]). in multivariable propensity score-adjusted analyses there was a statistically significant association with having a mother with atopic asthma compared with having a mother without asthma for infants with hrv-induced lrtis compared with rsv-induced lrtis (adjusted odds ratio, . ; % ci, . - . ). there was not a statistically significant difference between infants having a mother with nonatopic asthma compared with no asthma for infants with hrv-induced lrtis compared with those with rsv-induced lrtis (adjusted odds ratio, . ; % ci, . - . ) . in separate analyses for infants with either hrv or rsv infection, we examined whether the severity of the infant's respiratory tract infection was associated with having a mother with self-reported asthma. in infants with hrv, the association between self-reported maternal asthma and infant infection severity was not statistically significant in the ari group (both urtis and lrtis; adjusted odds ratio, . ; % ci, . - . ) or the lrti group (adjusted odds ratio, . ; % ci, . - . ; table ii ). in infants with rsv, the association between self-reported maternal asthma and infant infection severity was not significant in the total ari group (adjusted odds ratio, . ; % ci, . - . ) or the lrti group (adjusted odds ratio, . ; % ci, . - . ; table ii ). next, in separate analyses for infants with either hrv or rsv infections, we examined whether the severity of the infant's respiratory tract infection was associated with whether the infant's mother had atopic or nonatopic asthma. among infants with hrv-induced ari, having a mother with atopic asthma was associated with increased ari severity (fig ) , and in adjusted analyses there was a more than -fold increased relative odds of having more severe illness compared with infants whose mothers did not have asthma (adjusted odds ratio, . ; % ci, . - . ; table ii ). the interaction analysis investigating a differential effect of maternal atopic asthma by whether the infant had hrvor rsvon infection severity was also statistically significant (p . ). this relationship was not seen when limited to the subgroup of infants with hrv-induced lrtis; however, the number of infants with hrv-induced lrtis was very small (n ) and could be adjusted through propensity scores for only the infant's age, sex, and birth weight (table ii) . there was not an association between maternal atopic asthma and infection severity in infants with rsv-induced aris or the rsv-induced lrti subgroup (table ii) . rsv and hrv are the most common viruses associated with infant aris, and rsv-and hrv-induced lrtis are a leading cause of respiratory morbidity and hospitalizations in the first year of life. - viral lrtis during infancy and early childhood are also well established to be associated with an increased risk of asthma later in childhood. [ ] [ ] [ ] , because of the known differential risk of early childhood asthma after rsv-and hrv-induced infant infections, we were interested in studying whether a familial predisposition to asthma and allergies was associated with the viral cause of the infant's ari and the severity of the ari. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] several small studies have not found an association between familial predisposition and bronchiolitis; however, in our prior large, population-based cohort investigation of infants, we found that having a mother with asthma was associated with increased risk and severity of bronchiolitis during infancy. in the current investigation we sought to expand on previous findings by addressing the research questions of whether infants with hrvinduced aris or lrti subgroups are more likely to have a familial predisposition to asthma than those with rsv-induced infections and whether a familial predisposition to asthma is associated with more severe infant hrv-or rsv-induced aris. in our previous work we found that although, as a group, infants with a history of bronchiolitis had an increased risk of early childhood asthma, infants who had bronchiolitis during hrvpredominant months had a % increased risk of early childhood asthma compared with infants who had bronchiolitis during rsvpredominant months. furthermore, in the coast birth cohort, in which all children have a familial predisposition to asthma, , investigators found that hrv-induced wheezing illnesses in the first years of life were associated with a . -fold relative odds of asthma at years compared with a . -fold increase among children with rsv. we were able to assess whether maternal asthma and atopy were associated with the viral cause and the virus-specific severity of the infant's infection leading to an unscheduled health care visit because this cohort consisted of infants with and without a familial predisposition to asthma, and we used objective measures of maternal atopy and molecular techniques to determine the viral cause of the infant's respiratory tract infection. we found that infants with a mother with atopic asthma had an increased relative odds of having hrv-induced aris than rsv-induced aris compared with infants whose mothers did not have asthma. it is notable that although the prevalence of self-reported maternal asthma is higher in children with hrv ( %), the % prevalence of maternal asthma among infants with rsv-induced aris is higher than the asthma prevalence in the adult population in the united states. these study findings suggest that for infants with hrv-induced aris in particular, a familial predisposition to asthma might partly explain the increased risk of asthma seen later in life and/or that a familial predisposition to asthma predisposes infants to clinically significant hrvinduced aris during infancy, a viral infection that, unlike rsv, can often be isolated from young children without clinically obvious respiratory symptoms. we also found that infants with hrv who had a mother with atopic asthma had more than a -fold increased relative odds of having more severe illness than infants whose mothers did not have asthma. among those with rsv-induced aris, there was no relationship between maternal asthma and atopy and ari severity. the relationship between maternal atopic asthma and the infant's hrv infection severity is intriguing given the described altered host response to hrv, as has been found in vitro and among subjects with asthma. , [ ] [ ] [ ] [ ] [ ] these data support the notion of differential susceptibility to hrv among patients with atopic asthma. continued follow-up of these children until the age of years will further delineate whether it is the subset of infants with hrv-induced aris and a familial atopic predisposition who will have asthma and allergic diseases later in childhood or have more severe asthma or recurrent exacerbations. ultimately, this might help us to understand whether there is an altered host response or increased susceptibility to hrv among patients with atopic asthma. there are several limitations of this work. this study included a convenience sample of mother-infant dyads in which all infants presented for an unscheduled health care visit and not a cohort followed from birth. however, the study included participants recruited during viral seasons over years, which should serve to strengthen the generalizability of the findings. a single episode of rsv-or rhinovirus-induced ari was captured. it is likely that children had additional viral infections during infancy. the primary focus of this study was the association of a maternal atopic predisposition and the infant's ari severity, and therefore we were not able to investigate the outcome of bronchiolitis incidence as in our larger, population-based retrospective cohort of mother-infant dyads. in addition, maternal asthma was determined based on self-report and not based on objective criteria, such as airway reversibility testing. however, we used a validated instrument and self-reported asthma in young adults in whom there is little overlap with other diseases, and thus there is high specificity. furthermore, this study included objective measures of atopy. lastly, we cannot completely rule out the possible influence of unknown or unmeasured potential confounding factors, as with all observational studies. in summary, infant hrv infections requiring clinical care were more strongly associated with having a mother with atopic asthma than infant rsv infections. in addition, infants with hrv-induced aris who had a mother with atopic asthma had more than a -fold increased relative odds of having a more severe ari compared with that seen in infants with hrv whose mothers did not have asthma. this relationship was not seen in infants with rsv infections. these findings suggest that there is likely an underlying genetic basis for the risk of and response to respiratory tract infections during infancy and that the mechanisms underlying the increased asthma risk after hrv-and rsv-induced bronchiolitis might be different. future longitudinal investigations successful at preventing or modifying the host response to infant viral infections will provide insight into the relationship of infant viral infections and early childhood asthma, as will investigations that assess the atopic host and nonatopic host response to select respiratory pathogens. key messages d infants whose mothers had atopic asthma had increased relative odds of having a rhinovirus-induced ari than rsv infection compared with the odds in infants whose mothers did not have asthma. d in infants with rhinovirus, having a mother with atopic asthma was associated with -fold increased relative odds of more severe illness, a relationship not seen in infants with rsv. d for infants with rhinovirus-induced aris, a familial atopic predisposition might partly explain the subsequent increased risk of asthma and differential susceptibility to rhinovirus among asthmatic patients. bronchiolitis-associated hospitalizations among us children risk factors for respiratory syncytial virus-associated lower respiratory illnesses in the first year of life the increasing burden and risk factors for bronchiolitis-related medical visits in infants enrolled in a state healthcare insurance plan earlylife respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age the severity-dependent relationship of infant bronchiolitis on the risk and morbidity of early childhood asthma rhinovirus illnesses during infancy predict subsequent childhood wheezing the impact of respiratory viral infection on wheezing illnesses and asthma exacerbations atopy does not predispose to rsv bronchiolitis or postbronchiolitic wheezing wheezing, asthma, and pulmonary dysfunction years after infection with respiratory syncytial virus in infancy asthma and wheezing in the first six years of life. the group health medical associates respiratory syncytial virus in early life and risk of wheeze and allergy by age years viral infections, atopy, and asthma: is there a causal relationship? the childhood origins of asthma (coast) study childhood infections, the developing immune system, and the origins of asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease respiratory status and allergy nine to years after acute bronchiolitis family history of atopy and clinical course of rsv infection in ambulatory and hospitalized infants maternal asthma and maternal smoking are associated with increased risk of bronchiolitis during infancy host and viral factors associated with severity of human rhinovirusassociated infant respiratory tract illness wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the tennessee children's respiratory initiative: objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases relationship of maternal vitamin d level with maternal and infant respiratory disease international study of asthma and allergies in childhood (isaac): rationale and methods in vitro assays for the diagnosis of ige-mediated disorders in vitro diagnosis of atopic allergy in children. a comparison between total ige, conventional rast and a new multi rast (phadiatop) phadiatop compared to skin-prick test as a tool for diagnosing atopy in epidemiological studies in schoolchildren dexamethasone and salbutamol in the treatment of acute wheezing in infants prednisolone plus albuterol versus albuterol alone in mild to moderate bronchiolitis the central role of the propensity score in observational studies for causal effects r: a language and environment for statistical computing respiratory syncytial virus infection and recurrent wheeze/asthma in children under five years: an epidemiological survey community study of role of viral infections in exacerbations of asthma in - year old children the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings the september epidemic of asthma exacerbations in children: a search for etiology rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing allergic sensitization is associated with rhinovirus-, but not other virus-, induced wheezing in children season of infant bronchiolitis and estimates of subsequent risk and burden of early childhood asthma asthma prevalence, health care use, and mortality: united states asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma rhinoviruses in the pathogenesis of asthma: the bronchial epithelium as a major disease target rhinovirus infection induces expression of airway remodelling factors in vitro and in vivo validation of questionnaire and bronchial hyperresponsiveness against respiratory physician assessment in the diagnosis of asthma we conducted an analysis of mother-infant dyads enrolled in the tcri to investigate the association of a familial atopic predisposition with the viral key: cord- -dq y gkc authors: leung, ting fan; to, man yin; yeung, apple c.m.; wong, yun sze; wong, gary w.k.; chan, paul k.s. title: multiplex molecular detection of respiratory pathogens in children with asthma exacerbation date: - - journal: chest doi: . /chest. - sha: doc_id: cord_uid: dq y gkc background up to % of asthma exacerbations in white children are associated with viral upper respiratory infections. the relative importance of different respiratory pathogens and relevant microbiological data in asian children are unclear. this study elucidated the epidemiology of respiratory infections in hong kong children with asthma exacerbation. methods a total of children aged - years with asthma exacerbations and controls with stable asthma were recruited. the severity of asthma exacerbations was assessed according to global initiative for asthma guideline, and subjects aged years or older performed exhaled nitric oxide and spirometric measurements. nested multiplex polymerase chain reaction was used to detect different respiratory pathogens. results respiratory pathogens were detected in ( . %) subjects. the presence of any respiratory pathogen was associated with asthma exacerbation (odds ratio [or], . ; % ci, . – . ; p < . ). specifically, human rhinovirus (hrv) infection was more common among children with asthma exacerbation (or, . ; % ci, . – . ; p = . ). all other pathogens or coinfections were not associated with asthmatic attacks. none of these respiratory infections was associated with the severity of asthma exacerbation (p > . for all). during peak hrv season in the winter of to , this virus was detected in . % of children with asthma exacerbations. conclusions respiratory viral infections are commonly found in children with asthma exacerbation, with hrv being the most important pathogen in our patients. respiratory viral infection is a triggering factor for asthma exacerbation but does not correlate with its severity. years with a defi nitive diagnosis of asthma because it may be diffi cult to differentiate acute bronchiolitis from asthma in the younger children. patients who received antimicrobial agents (eg, neuraminidase inhibitors, ribavirin, and macrolides) within weeks before study were also excluded. the severity of asthma exacerbation was classifi ed according to global initiative for asthma guidelines. infection was defi ned as the detection of respiratory pathogens by our nested multiplex pcr assays. our primary outcome was the difference in detection rate for any respiratory pathogen between children with asthma with acute exacerbation and controls (ie, stable asthma). secondary outcomes consisted of differences in the clinical severity of asthma exacerbation, lung function parameters, and fractional exhaled nitric oxide concentration (feno) in relation to patients with different respiratory pathogens. the clinical research ethics committee of our university approved this study. following informed written consent, subjects had clinical assessment followed by feno and spirometric measurements. subjects years of age and older underwent online feno measurement using a chemiluminescence analyzer (sievers; boulder, co) according to international guidelines. the mean feno of three no plateau values was recorded. feno was measured within h of hospitalization for children with acute asthma and at the clinic visit for stable patients. the former group was allowed to commence systemic corticosteroids as clinically indicated prior to feno because it would be unethical to withhold such treatment until this study. following feno, they performed spirometry (compact ii; vitalograph; buckingham, uk ) to measure fev , fvc, and fev /fvc. in accordance with local infection control policy, nasopharyngeal aspirates (npas) were collected in negative-pressure isolation rooms. deep nasal swabs were obtained as an alternative in situation where an isolation facility was unavailable. these specimens were put immediately in viral transport medium and kept at °c during transportation. both viral rna and dna were extracted on the same day of collection by purelink viral rna/ dna mini kit (invitrogen; carlsbad, ca). rna extracted was converted to cdna by reverse transcriptase (superscript iii reverse transcriptase; invitrogen). all dna and cdna were used immediately for nested multiplex pcr for respiratory pathogens as described previously. tables a and b in the online supplement summarize sequences and amplicon sizes of the outer and inner sets of pcr primers. briefl y, each nested multiplex pcr assay detected four pathogens. group comprised infl uenza a and b group-specifi c and subtypes h n , h n , h n specifi c primers; group comprised parainfl uenza viruses (piv- , piv- , piv- , piv- a, and piv- b); group comprised rsv a and b, hrv, and enterovirus; group comprised hcov-oc , hcov- e, sars-cov, and hmpv; and group comprised m pneumoniae , c pneumoniae , hbov, and adenovirus. both the fi rst and second rounds of pcr were conducted in -m l reaction mixtures using fast thermal cycler (applied biosystems; foster city, ca). two microliters of cdna was used as the template for the fi rst round of pcr for groups to , whereas m l of the extracted preparation was used for group . in the second round of pcr, a . -m l aliquot of the fi rst-round pcr product was used as a template. table c in the online supplement summarizes the pcr conditions of fi ve multiplex nested pcr assays. the pcr products were stained by sybr safe (invitrogen) and visualized by electrophoresis in . % agarose gels. four corresponding positive controls and one negative control (sterile water) sample were respiratory viruses, such as human coronavirus (hcov)- e, hcov-oc , and human metapneumovirus (hmpv), are common causes of upper rti, [ ] [ ] [ ] but their relation to asthma exacerbation remains u nclear. allander et al developed a system for largescale molecular virus screening of clinical samples based on host dna depletion, random polymerase chain reaction (pcr) amplifi cation, large-scale sequencing, and bioinformatics. a more recently described parvovirus called human bocavirus (hbov) was identifi ed in children with lower rtis, but its relation to asthma exacerbation is uncertain at present. asthma exacerbation may also be caused by atypical bacteria. chlamydophila pneumoniae , an obligate intracellular respiratory pathogen, was linked to asthma exacerbation in children. among children hospitalized for wheezing, johnston detected respiratory viruses in % of patients aged less than months and % in those aged more than years. asthma-related symptoms resolved or signifi cantly improved in half of adult patients with asthma seropositive for c pneumoniae who were treated with macrolides or doxycycline. mycoplasma pneumoniae is detected by pcr in more than % of patients with asthma, but it was also found in % of upper airway secretions from patients with stable asthma. it is important to understand the roles different respiratory pathogens play in precipitating asthmatic attacks in order to determine ways to reduce the health-care burden associated with asthma-related hospitalization. the objectives of this study were: ( ) to investigate the importance of different respiratory patho gens in childhood asthma exacerbation, and ( ) to delineate the epidemiology of respiratory pathogens causing asthma exacerbation in hong kong children. this study recruited children with asthma aged - years with disease exacerbation who received treatments either in pediatric wards or outpatient clinics of a university teaching hospital between january and february . inpatients hospitalized for asthma exacerbation were assessed within h of hospitalization. age-and sex-matched children with asthma who were free from rtis for weeks or longer and were seen in our allergy clinic within the same week as the above children with acute asthma were recruited as controls. asthma diagnosis was made according to british thoracic society criteria. briefl y, older patients were either hyperresponsive to methacholine or showed reversible airfl ow limitation, whereas young children with asthma had three or more episodes of cough, shortness of breath, and wheezing during the months before the study. these young patients also showed good response to bronchodilator. this study selected only children older than included for each group simultaneously. in order to prevent pcr contamination, reagent preparation, sample processing, and nested pcr assays were performed in separate rooms away from where amplifi ed products were analyzed. aerosol-resistant pipette tips were used throughout the experiments. as rtis are age-dependent, we tried to match one control per patient with respect to their age and sex. however, we failed to recruit this target number of controls because many children with stable asthma had rti symptoms during winter. the detection rates for respiratory pathogens between cases and controls were analyzed by x or fisher exact test. the severity of asthma exacerbation, feno, and spirometric parameters were analyzed between subgroups with different pathogens by x or student t test. multivariate logistic regression was used to identify respiratory pathogens associated with asthma exacerbation, adjusted for age, inhaled corticosteroid (ics) treatment, and domestic tobacco smoke exposure as covariates. all analyses were performed twotailed using spss v. (spss inc.; chicago, il), with the level of signifi cance set at . . two hundred nine children with asthma exacerbation, including patients hospitalized in our pediatric wards and six who attended our outpatient clinics, and controls with stable asthma were recruited. table shows the characteristics of these patients. children with asthma exacerbation were younger than the controls, mainly because of our inability to recruit one age-matched control for each child with asthma exacerbation. similar proportions of patients in the two groups received regular ics treatment. suffi cient respiratory samples were collected from ( . %) cases and all controls; these consisted of npa samples and nasal swabs. respiratory pathogens were detected in ( . %) subjects. table summarizes the distributions of respiratory pathogens in two groups of patients. the presence of any virus with or without atypical bacteria was associated with asthma exacerbation ( p , . for both). specifi cally, hrv infection was more common among children with asthma exacerbation (odds ratio [or], . ; % ci, . - . ; p . ). on logistic regression, asthma exacerbation was associated with the detection of hrv (or, . ; % ci, . - . ; p . ), any respiratory virus (or, . ; % ci, . - . ; p . ), or any respiratory pathogen (or, . ; % ci, . - . ; p . ). none of the respiratory pathogens or their coinfection was associated with the severity of asthma exacerbation ( p . . for all). age, gender, and ics treatment did not affect the detection of respiratory pathogens in patients with asthma exacerbation ( p . . for all). table summarizes the clinical features of patients with asthma exacerbation in relation to hrv infections. respiratory pathogens were detected in % of these patients, and hrv peaked in winter and early spring. these multiplex nested pcr assays were specifi c and -to , -fold more sensitive than conventional methods in detecting the viruses. our assays detected Յ nucleic acid copies for all viruses (except enteroviruses), which were comparable to those reported in widely quoted highthroughput multiplex pcr assays. specifi cally, our nested pcr was able to detect one cdna copy of hrv. the present study used the same method, except for legionella pneumophila being replaced by hbov, to investigate the infective causes of asthma exacerbation in hong kong children. hrv infections also peaked in winter of / in children with asthma exacerbation ( fig ) . our detection rate ( %) was similar to previous local studies , but lower than those published in white populations. it is uncertain whether our low hrv detection rate was due to limitations of the pcr technique, which is less likely in view of our previously noted in vitro results, or a genuinely low incidence of hrv infection in hong kong children. further studies in other asian populations are needed to confi rm our fi ndings. hrv infection was associated with asthma exacerbation in the children, which is consistent with feno was the only parameter that differed between patients with and without hrv, being signifi cantly lower in the former group ( p . ). ten controls were hrv positive, and mean (sd) feno of those with and without hrv were . ( . ) ppb and . ( . ) ppb, respectively ( p , . ). table summarizes the relationship between age and different respiratory pathogens in patients with asthma exacerbation. patients with asthma exacerbation caused by respiratory viruses were younger than those without identifi able viral infections ( p , . ). this fi nding was attributed mainly to rsv ( p , . ) and infl uenza a and hmpv infections ( p , . for both). age did not differ between the case and control groups with infections by other organisms, including hrv, or with coinfections. figure illustrates the seasonal pattern of hrv, which was found in patients throughout the study period ( Ն %) and peaked in winter (november-december) of to . similarly, . % of controls had hrv in autumn-winter (september-october), but none of them were hrv positive in spring-summer (march-august). the low positive rates for other respiratory pathogens in our subjects preclude our analysis of their seasonality patterns. b. on the other hand, hrv infection was detected in % of our subjects with stable asthma who did not experience any symptom or sign of disease exacerbation. in a longitudinal study of healthy children, . % of all hrv infections were asymptomatic. future studies should delineate the pathogenic linkage between hrv and worsened asthma. during the past few years, there has been impressive advance in our understanding of the interactions between hrv and host immunity. hrv infects human cells via ligation with its major group receptor intercellular adhesion molecule . infected respiratory epithelial cells, and possibly macrophages, produce a variety of proinfl ammatory cytokines, chemokines, and leukotrienes. these mediators in turn attract different infl ammatory cells to the airway, resulting in worsened immunopathology and increased bronchial hyperresponsiveness observed in patients with asthma. in addition, airway epithelium from patients with asthma is defi cient in mounting adequate antiviral responses to hrv. , hbov was detected in . % of , local children hospitalized for acute rtis, and seasonal distribution was noted from september to february. despite this, the detection of hbov in . % of cases and . % of controls was not associated with asthma exacerbation in the present study. this fi nding might be explained by our exclusion of infants and young children, who were at increased risk of hbov infection. , three-fi fths of adults with asthma exacerbation had m pneumoniae and/or c pneumoniae , and telithromycin was shown to be a useful treatment in these patients. m pneumoniae was detected in more than half of patients with asthma, and also in upper airway secretions from % of patients with chronic stable asthma. hahn claimed oral macrolides to be effi cacious for patients with acute asthma. on the other hand, cunningham et al failed to show any relation between m pneumoniae and childhood asthma exacerbation. m pneumoniae and c pneumoniae were detected only in . % of our children with asthma exacerbations, which was similar to that observed in patients with stable asthma. our fi ndings do not support atypical bacteria to be important pathogens for asthma exacerbations in children or the usefulness of macrolides in treating these patients. the major limitation of this project relates to its study power. the number of controls was much lower than that of our recruited cases, mainly because casecontrol matching within week was not possible on many occasions when stable and especially younger patients also complained of nonspecifi c upper respiratory symptoms (eg, rhinorrhea, blocked nose, sore throat) during change of weather. our sample size had a power of % for detecting any difference in the detection of any virus between cases and controls, published data about the importance of hrv in white populations. in the childhood origins of asthma birth cohort, a total of children were followed prospectively from birth to years of age. hrv-associated wheezing in years and were the strongest predictor for asthma diagnosis at the age of years. nearly % of children who wheezed with hrv in year subsequently developed asthma. two other studies found hrv to be the most important microbiological risk factors for asthma diagnosis and disease exacerbation. [ ] [ ] [ ] more recently, miller et al reported that childhood asthma exacerbations were associated with the novel group c of hrv rather than the two previously known phylogenetic groups a and results expressed in mean (sd) and only included data for pathogens that were detected in fi ve or more patients. a p , . for between-group comparisons. b p , . for between-group comparisons. c p , . for between-group comparisons. dr g. w. k. wong: contributed to subject recruitment and manuscript preparation. dr chan: contributed to designing and supervising virologic investigations and participated in manuscript preparation. financial/nonfi nancial disclosures: the authors have reported to chest that no potential confl icts of interest exist with any companies/organizations whose products or services may be discussed in this article. but had a marginal power of % for hrv infection (graphpad statmate; san diego, ca) and , % for the detection of other respiratory pathogens because of their rarity. thus, larger studies are needed to delineate the possible association between asthma exacerbation and rtis by these organisms. the lack of standardization on the methodology of hrv detection would also pose a problem. a recent study revealed that hrvs consist of . distinct serotypes. in view of this degree of phylogenetic heterogeneity, the pcr primers designed for our multiplex assays were not able to detect all hrvs. despite the use of sensitive multiplex assays as discussed previously, our molecular approach for detecting viruses would miss some hrvs that were not covered by our pcr primers. future studies need to adopt multiple pcr primers that specifi cally target as many hrv serotypes as possible. another weakness is that npa samples were collected from ( . %) subjects, whereas nasal swabs were collected from the remaining subjects. as we previously reported, the overall sensitivity of detecting infl uenza, parainfl uenza, rsv, and adenovirus in npa was higher than that obtained by nasal swabs in local children. on the other hand, we did not have relevant data for hrv. although more than % of subjects had npa samples, it is possible that we might have missed some organisms in those with only nasal swabs. we also observed that only % of patients hospitalized for asthma exacerbation had successfully performed feno measurement according to guideline (table ) . as patients with severe bronchospasm were probably too breathless for the procedure, only patients with milder attacks would contribute to feno readings in this group. in addition, a substantial proportion of these patients were treated with systemic corticosteroids prior to feno. these reasons explain the lower feno in these patients when compared with patients with stable asthma. in conclusion, respiratory viruses and atypical bacteria are detected in more than half of hong kong children with asthma exacerbation. hrv infection is the most important risk factor for asthma exacerbation in these patients. nonetheless, none of these pathogens is associated with severity of asthma exacerbation. prevalence of respiratory and atopic disorders in chinese schoolchildren assessing disease burden of respiratory disorders in hong kong children with hospital discharge data and linked laboratory data community study of role of viral infections in exacerbations of asthma in - year old children role of viruses and atypical bacteria in exacerbations of asthma in hospitalized children: a prospective study in the nord-pas de calais region (france) the burden of infl uenza illness in children with asthma and other chronic medical conditions a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl in children hospitalized with respiratory tract infections in belgium human metapneumovirus detection in patients with severe acute respiratory syndrome children with respiratory disease associated with metapneumovirus in hong kong cloning of a human parvovirus by molecular screening of respiratory tract samples chronic chlamydia pneumoniae infection and asthma exacerbations in children infl uence of viral and bacterial respiratory infections on exacerbations and symptom severity in childhood asthma treatment of chlamydia pneumoniae infection in adult asthma: a before-after trial detection of mycoplasma pneumoniae in the airways of adults with chronic asthma a link between chronic asthma and chronic infection scottish intercollegiate guidelines network (sign) the group health medical associates . asthma and wheezing in the fi rst six years of life global initiative for asthma . bethesda, md : national heart, lung, and blood institute measurement of exhaled nitric oxide in children comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of infl uenza rapid multiplex nested pcr for detection of respiratory viruses high-throughput, sensitive, and accurate multiplex pcr-microsphere fl ow cytometry system for large-scale comprehensive detection of respiratory viruses identifi cation of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays asthma exacerbations. : aetiology wheezing rhinovirus illnesses in early life predict asthma development in high-risk children prevalence of viral respiratory tract infections in children with asthma rhinovirus infection preferentially increases lower airway responsiveness in allergic subjects the relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking new vaccine surveillance network . a novel group of rhinoviruses is associated with asthma hospitalizations picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season host immune responses to rhinovirus: mechanisms in asthma asthmatic bronchial epithelial cells have a defi cient innate immune response to infection with rhinovirus role of deficient type iii interferon-lambda production in asthma exacerbations pediatric hospitalization of acute respiratory tract infections with human bocavirus in hong kong the effect of telithromycin in acute exacerbations of asthma sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection key: cord- - fmsxx s authors: goffard, anne; lambert, valérie; salleron, julia; herwegh, stéphanie; engelmann, ilka; pinel, claudine; pin, isabelle; perrez, thierry; prévotat, anne; dewilde, anny; delhaes, laurence title: virus and cystic fibrosis: rhinoviruses are associated with exacerbations in adult patients() date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: fmsxx s background: few studies have suggested the potential role of respiratory viruses in cystic fibrosis (cf) exacerbation, but their real impact is probably underestimated. method: sixty-four sputum samples collected from adult patients were included in the study: samples were collected during exacerbation of cf, and during the stable phase. after extraction, nucleic acids were tested for the presence of respiratory viruses. when rhinovirus (hrv) was detected, the ′utr and vp / regions were sequenced, and phylogenetically analyzed. the characteristics of patients in exacerbation and stable phase were compared. results: viruses were found in % of samples. the hrv viruses were the most frequently detected followed by coronaviruses. only the hrv detection was significantly associated with the occurrence of cf pulmonary exacerbation (p < . ). characterization of ′utr and vp / regions of the hrv genome specified that hrv-a, -b, -c were detected. all hrv-c were recombinant hrv-ca. conclusions: hrv were the most frequently detected viruses; their detection was significantly associated with the occurrence of an exacerbation. the reality of viral recombination between hrv was demonstrated in cf patients for the first time, raising the role of viruses in lung microbiota. further studies are now warranted to decipher virus impact in cf. cystic fibrosis (cf) is the major genetic inherited disease in the european caucasian population. in addition to bacteria, which are well known to cause recurrent exacerbations of cf-associated pulmonary disease and often determine the vital prognosis of patients [ ], many airborne particles such as viral entities or fungal spores can also colonize the respiratory tract of patients [ , ] . for a long time, the detection of respiratory viruses, especially in exacerbations of chronic lung diseases has been underestimated because of the lack of sensitive methods for virus detection. since the sensitive molecular methods for detection of viruses are more and more common, several recent studies highlight the clinical importance of respiratory viruses especially during exacerbation of asthma, chronic obstructive pulmonary disease (copd) or cf [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, viruses that were not previously sought are now detected, such as human rhinoviruses (hrvs) within the picornaviridae family. among the hrv genus, a new species of viruses named hrv-c has been described recently, is involved in severe respiratory infections in elderly and in immunocompromised patients [ , [ ] [ ] [ ] hrv-c has been described in patients with cf exacerbation but the real impact in the progression of the lung disease remains unclear [ ] . the primary objective of our study was to investigate the frequency and species of viruses present in sputa collected from cf adult patients with either pulmonary exacerbation or stable disease, and to determine the clinical relevance of viruses. as the most frequently detected viruses were hrvs, we phylogenetically characterized them and identified potential clinical associations, expressly for a significant relationship between hrv detections and occurrence of cf exacerbations. sixty four sputum samples collected from cf patients in lille ( samples, patients), dunkerque ( samples, patients), and grenoble ( samples, patients) between october and december were prospectively screened for respiratory virus carriages (reference number of the institutional ethics committees of lille hospital, cpp / -written informed consent was obtained from each patient). all patients had a well-documented diagnosis of cf with either the mutations identified in the cftr gene or an abnormally high sweat chloride test. they were included by physicians according to the same criterion (an annual check-up or an exacerbation situation that required an expectorated sputum sample that allowed us to search also for bacterial and fungal microorganisms). clinical data including spirometry, therapeutic, radiological and biological data were collected by clinic staff at each time of the visit. at each sampling time, the clinical status of the patients was defined as pulmonary exacerbation or stable period according to the physician defined requirement (i.e. recent changes in clinical parameters and/or modification of the pulmonary function provide criteria of exacerbation according to ers statement) [ ] . twenty eight patients had one sputum examined, and patients had two sequential sputa examined, which were considered as independent events since the delay between the samples was at least months [ , ] . samples were performed by expectoration into a sterile cup after a water rinse to prevent excessive salivary contamination, analyzed according to a standardized protocol as previously described, and screened for respiratory virus carriage [ ] . total nucleic acids were obtained after mechanical lysis of sputum sample. briefly, l of each sample were subjected to fluidification in a magnalyser (roche diagnostics) according to the manufacturer's instructions. qiaampmin elute virus spin kit (qiagen) was used on crude lysate according to the manufacturer's instructions. purified nucleic acids were frozen at − • c until use. reverse transcription was performed with revertaid first strand cdna synthesis kit (fermentas) using l of previously obtained rna. detection of respiratory viruses was performed with seeplex rv ace detection kit (seegene) in three independent pcr reactions targeting influenza viruses a and b, rsv a and b, human adenovirus, human metapneumovirus (hmpv), human coronavirus (hcov e, nl , oc and hku ), human parainfluenza viruses - , hrv, human enteroviruses, and human bocaviruses in three independent pcr reactions. amplification products were visualized after semi-automated capillary electrophoresis in a tapestation lab (eurobio) and analysis was performed with tape ds software. samples that were positive for hrv were identified, based on sequencing of the vp /vp and utr regions, according to the previously described nested rt-pcr strategy and the primers listed in table [ ] . the three hrv-specific vp /vp , utr inner sense primers were included in the second amplification step. the amplicons were directly sequenced using abi prism xl (life technologies) and results were analyzed with the seqscape . software (applied biosystem). phylogenetic trees of the vp /vp and utr regions were constructed with unambiguously aligned sequences ( and sites for vp /vp and utr, respectively) using neighbor joining of pairwise maximum composite likelihood method implemented in mega program [ ] . the reliability of internal branches was assessed using the bootstrap method with replicates. accession numbers of the sequences included in the dataset are listed in fig. . quantitative variables were described by median, mean and standard deviation; qualitative variables were described by frequency and percentage. the comparison of sub-population regarding hrv carriage were performed using the mann-whitney u test for quantitative variables. qualitative variables were compared using chi-square test or fisher's exact test when appropriate. a p value of < . was considered significant. all statistical analyses were performed under sas software (sas institute, cary, nc, usa; version . ). interactions between rhinovirus infection and the different variables were investigated with one interaction term with respective subterms in the model (univariate model) and then repeated with adjustments to dyspnea, s-k score, abpa, segmental or sub-segmental bronchiectasis, fungus detection, intravenous antibiotics or azithromycin treatment or systemic steroids regimens (multivariate model). forty six patients ( males and females) were included in this study, with a mean age of years (mean ± sd: ± . ). their characteristics at enrolment and at each sampling time are summarized in table . a majority of patients ( %) were homozygous or heterozygous for the del-f mutation in the cftr gene overall, viruses were identified from out of sputum samples ( %). rhinoviruses ( / ; %) and hcov ( / ; %) were the main identified agents. eight hrvs were isolated from patients with acute exacerbation ( / occurrences; %, fig. a ) and from a patient during a stable state ( / episodes; . %, fig. b ). two ( / ; %) oc /hku or e/nl hcovs have been detected from sputa collected during pulmonary exacerbation (fig. a) . three ( / ; %) hcovs, ( / ; %) influenza a virus and ( / ; %) parainfluenza virus (piv- ) were detected in sputa collected from patient in stable clinical condition (fig. b) . detection of respiratory viruses occurred mainly in the winter season (fig. c) . no significant differences were found in baseline values between the subgroups composed of patients with and without virus detection. viral respiratory infections in our population of cf patients were not significantly associated with either respiratory symptoms, or deterioration of body mass index, of shwachman-kulczycki score, and of spirometry parameters, or comorbidity factors, or microbial colonization, or an increased use of antibiotics, of antifungal or steroids ( table ). alterations confirmed by radiology or scanner as proximal bronchiectasis or as segmental/sub-segmental bronchiectasis (identified in . % and . % of the population, respectively) were not significantly associated with virus detection, but missing data were about %. regarding hrv detection, neither general features of cf patients (such as cftr mutations or body mass index), nor spirometric values, nor clinical criteria of respiratory function (such as dyspnea, wheezing, ronchi, or crackles), nor radiological and microbial parameters were significantly associated with hrv detection ( table ). regarding clinical criteria of respiratory function, missing data were about % for dyspnea, and . % for wheezing, ronchi, and crackles, which might explained the absence of statistical significance. interestingly, detection of rhinoviruses was significantly associated with pulmonary exacerbation (p = . , table ). sequencing the vp /vp region (nucleotide positions - ) was successfully performed in the hrv positive samples. the obtained sequences were aligned with published complete genome sequences and available partial vp /vp sequences ( fig. a) . four hrv sequences were identified as belonging to hrv-a species, to hrv-c species and to hrv-b species. the samples that were assigned to hrv-c or hrv-a species were characterized further by sequencing the utr region (nucleotide position - ; fig. b ). using a bootstrap value of % or greater to define clades, topologies of the trees constructed from vp /vp and utr regions were in agreement with previous published results [ ] [ ] [ ] . surprisingly, the sequences obtained classified as hrv-c by comparative analysis of vp /vp sequences were related more closely to hrv-a strains when their utr regions were analyzed. all query sequences grouped within the hrv-a clade, with bootstrap value of and % (figure ) , and belong to hrv-ca variants, according to recent published data [ ] [ ] [ ] [ ] . in the present study, we reported detection of respiratory viruses from adult patients with cf during either routine visit or acute pulmonary exacerbation. we detected respiratory viruses in % of sputum samples. this proportion is close to previously published results obtained using conventional diagnosis methods but notably smaller than other recently published data, which might be related to our sampling method [ , , [ ] [ ] [ ] . upper respiratory tract sample such as nasal washes or oropharyngeal swabs are the most frequent sample types used to detect respiratory viruses. sputa were considered as lower respiratory tract samples and has been recently described as suitable to identify viruses in cf patients [ , ] . here, we performed a mechanical pre-treatment of each sputum sample before nucleic acid extraction; this pretreatment is newly recommended for respiratory virus detection in sputa from patients with asthma or cf [ , ] . moreover, sputum analysis has been used for rhinovirus detection in cf patients, who present exacerbation and sputum production [ ] . even if there is only a little data on the incidence of viral pathogens causing exacerbation in adult cf patients, coronavirus, piv- , pandemic a/h n were detected in our population as previously reported [ , , , [ ] [ ] [ ] . our screening for respiratory viruses showed the presence of coronavirus in samples collected both from patients with exacerbation and from patients during stable disease. however, the presence of coronavirus in samples is not associated with the occurrence of an exacerbation, according to previous study [ , ] . we detected influenza a virus in only one case, and therefore did not confirm the link between influenza infection and the occurrence of an exacerbation in cf previously described [ ] . due to the potential severity of influenza infection in cf, it is recommended to vaccinate patients against influenza viruses, but the patient positive to a/h n detection did not follow this recommendation and was not vaccinated. the low influenza a virus detection frequency observed here might be linked to the good matching of the influenza vaccine to the circulating influenza viruses during our study period, even if the vaccination status of our cf patient cohort was not documented. consistent with previous studies, we detected coronaviruses preferentially during the cooler months, hrv-a mainly in spring and autumn, and hrv-c variants preferentially during the autumn and winter months [ , , ] . hrvs were the most commonly detected viruses, as previously reported in cf children [ , , ] . whereas the physiopathology of virus-induced exacerbation in cf remains unclear, we established a significant association between hrv detection and respiratory exacerbation, confirming the results of recent studies [ , , ] . the detection of hrvs is more frequent in nasal swabs, especially in patients in a stable phase of cf [ ] . however, our results show that the detection of hrv in sputum is associated with the occurrence of an exacerbation suggesting that the analysis of sputum could be more relevant than nasal swabs when a viral cause of pulmonary exacerbation is suspected. some recent studies have shown that hrv-c are involved in severe respiratory infections especially during exacerbations of asthma [ , ] . numerous findings underscore the polymicrobial nature of respiratory tract as well as the importance of lung microbiota as a distinctive feature of tissue compartmentalization, but viral microbiota (or virome) has been poorly studied in respiratory tract [ , ] . respiratory virome appears composed of three major virus families: paramyxoviridae, orthomyxoviridae or picornaviridae for which a novel type of rhinovirus c was identified [ ] . here, we performed sequence-based typing of hrvs resulting in hrv-b, hrv-a, and hrv-c. as previously described, hrv-a and hrv-c were the most frequently detected viruses compared to the frequency of hrv-b detection [ ] . recent studies have shown recombination between the utr region of hrv-c genome and the coding region of hrv-a genome leading the authors to propose a new classification of hrvs in two subspecies, hrv-ca and -cc [ , , , ] . occurrence of such recombination was then confirmed, hrv-ca and hrv-cc being detected in various upper respiratory diseases [ ] . here, we identified all the hrv-c isolates as belonged to the sub-species hrv-ca by sequencing both the vp /vp and utr regions. to our knowledge, the present study describes for the first time the presence of hrv-ca subtype in patients with cf. although the clinical significance of such recombination is still matter of debate, our results confirm the well-known recombination ability of enteroviruses (notably hrvs); a fact of particular relevance when phage community represents a key point in the lung microbiota analysis as well as in the improvement of resistosome concept [ , , , ] . in fact, human microbiota represents a huge reservoir of mobilizable genes, as proposed by rolain and co-workers [ ] ; some of those genes encoding antimicrobial resistance that the authors named 'resistome' [ ] . this concept is based on few metagenomic studies that have analyzed viral and bacterial communities in cf sputum samples, revealing the presence of an important bacteriophage community containing genes encoding antimicrobial resistance, and demonstrating these bacteriophages are consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in cf patients [ , , , ] . to conclude, we have reported a relatively high frequency of respiratory viruses in a cohort of cf adult patients from france, and demonstrated for the first time that rhinovirus detection including newly identified hrv-ca variants are the most frequent and significantly associated with respiratory exacerbations. our results can be considered as contribution to the characterization the role of viruses in cf pulmonary exacerbation, underscoring the importance of the lung virome that remains poorly understood in the context of cf. the authors thank phrc -vaincre la mucoviscidose -pfizer laboratories for their financial support. a polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients the airway microbiota in cystic fibrosis: a complex fungal and bacterial communityimplications for therapeutic management inflammatory responses to individual microorganisms in the lungs of children with cystic fibrosis viruses exacerbating chronic pulmonary disease: the role of immune modulation viral infections in exacerbations of asthma and chronic obstructive pulmonary disease the role of respiratory viruses in cystic fibrosis rhinovirus c and respiratory exacerbations in children with cystic fibrosis incidence of viral respiratory pathogens causing exacerbations in adult cystic fibrosis patients detection of viral and bacterial respiratory pathogens in patients with cystic fibrosis clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia pulmonary exacerbation: towards a definition for use in clinical trials, report from the eurocarecf working group on outcome parameters in clinical trials dynamic colonisation by different pneumocystis jirovecii genotypes in cystic fibrosis patients screening respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp -vp typing reveals high incidence and genetic diversity of hrv species c mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods clinical associations and prevalence of scedosporium spp. in australian cystic fibrosis patients: identification of novel risk factors? allergic bronchopulmonary aspergillosis in cystic fibrosis -state of the art: cystic fibrosis foundation consensus conference evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection analysis of genetic diversity and sites of recombination in human rhinovirus species c genetics, recombination and clinical features of human rhinovirus species c (hrv-c) infections; interactions of hrv-c with other respiratory viruses characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing severe viral respiratory infections in infants with cystic fibrosis viral and atypical bacterial infections in the outpatient pediatric cystic fibrosis clinic role of respiratory viruses in pulmonary exacerbations in children with cystic fibrosis respiratory viruses in children with cystic fibrosis: viral detection and clinical findings metagenomics and metatranscriptomics: windows on cf-associated viral and microbial communities association of respiratory viral infections with pulmonary deterioration in patients with cystic fibrosis influenza-associated cystic fibrosis pulmonary exacerbations the differential clinical impact of human coronavirus species in children with cystic fibrosis human coronavirus nl infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong molecular epidemiology and dual serotype specificity detection of echovirus strains in finland a retrospective overview of enterovirus infection diagnosis and molecular epidemiology in the public hospitals of marseille high rhinovirus burden in lower airways of children with cystic fibrosis alveolar macrophages and cc chemokines are increased in children with cystic fibrosis metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals global distribution of novel rhinovirus genotype the abcs of rhinoviruses, wheezing, and asthma bacteriophages and diffusion of genes encoding antimicrobial resistance in cystic fibrosis sputum microbiota the authors thank all the clinicians from the three hospitals for their cooperation in collecting sputum samples and clinical data. the authors would like to thank carolyn engel-gautier for english editing. none. reference number of the institutional ethics committees of lille hospital, cpp / . written informed consent was obtained from each patient. key: cord- -ce wa authors: wang, zheng; malanoski, anthony p; lin, baochuan; kidd, carolyn; long, nina c; blaney, kate m; thach, dzung c; tibbetts, clark; stenger, david a title: resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: ce wa background: febrile respiratory illness (fri) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. a particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (hrv) and enteroviruses (hev) which are frequent causes of fri. resequencing pathogen microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking. results: using hrv and hev as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. a minimal number of probe sequences ( for hrv and for hev), which were potentially capable of detecting all serotypes of hrv and hev, were determined and implemented on the resequencing pathogen microarray rpm-flu v. / (tessarae rpm-flu). the specificities of designed probes were validated using hrv and hev strains. all strains were successfully detected and identified at least to species level. hrv strains and hev strains could be further differentiated to serotype level. conclusion: this study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse rna viruses with a minimal number of prototype sequences. the results demonstrated that the newly designed rpm-flu v. / can provide comprehensive and specific analysis of hrv and hev samples which implicates that this design strategy will be applicable for other genetically diverse viruses. human febrile respiratory illness (fri) results in significant annual health and economic burden worldwide, but the diversity and number of pathogens make differential diagnosis very challenging. thus, it represents a useful example where many organisms ranging from bacteria (haemophilus influenzae) to fairly conserved viruses (respiratory syncytial virus) to genetically diverse viruses, i.e. influenza a virus, human rhinoviruses (hrv), and human enteroviruses (hev) need to be detected for successful differential diagnosis. several technologies, mass-code™ multiplex rt-pcr system [ ] , electrospray ionization mass spectrometry analysis of pcr amplicons [ ] , luminex ® xmap™ [ ] , and various microarray-based approaches [ ] [ ] [ ] [ ] [ ] , are currently under development as diagnostic platforms to effectively and simultaneously detect and identify large numbers of diverse viral and bacterial respiratory pathogens. one high-density resequencing microarray platform, the respiratory pathogen microarray version (rpm v. ) , has been successfully demonstrated to identify a much broader range of pathogens (including bacteria and dna and rna viruses) in a single test at sensitivities and specificities that are similar to or improved over those of other technologies [ , ] . in addition, the rpm v. platform has the demonstrated capability to discriminate among known and previously unknown strains and variants of targeted pathogens [ , ] . while promising, the rpm v. platform was a proof-ofconcept microarray for the detection of common respiratory pathogens primarily encountered among military basic trainees. it did not provide comprehensive coverage of all potential respiratory pathogens and the design methodology used was not appropriate for genetically diverse viruses. the design methodology for the rpm v. microarray consisted of applying selection rules developed for long oligonucleotide microarrays. these rules were not optimal but worked for bacterial organisms and fairly conserved viruses since previous studies had shown a single sequence on a resequencing microarray could reliably detect and serotype strains with as much as to % variation [ , [ ] [ ] [ ] . their application to cover more diverse viral organisms was less successful. for example, the ' untranslated region ( 'utr) sequence chosen for hrv on the rpm v. only provided identification of the prototype hrv- and very little coverage of other hrv serotypes. the 'utr sequences, which are relatively conserved among hrv and hev, have been used in pcr and de novo sequencing for tentative viral identification or serotype classification in lieu of the much more variable capsid proteins that actually determine serotypes [ , ] . however, the 'utr sequences still have ~ to % nucleotide sequence variations among different serotypes so require more than one prototype sequence for proper identification and serotyping. serotyping hrv and hev is important to fri differential diagnosis because even though these "common cold" viruses generally only induce mild symptoms, they can cause a wide variety of other severe illnesses, such as aseptic meningitis [ ] , bronchitis and asthma [ ] . new resequencing pathogen microarray designs, versions . and . (rpm-flu v. / ), have been constructed to address the shortcomings of the previous design. the use of μm feature allows microarrays with greater coverage, currently , of common respiratory organisms and high human health risk zoonotic pathogens (bacteria and viruses). a new approach to select a minimal number of prototype sequences that can be used to detect all and correctly identify many of the relevant strains of genetically diverse viruses such as hrv and hev was developed. due to the great genetic diversity of hrv and hev, in order to ensure that designed probes (referred to as probe sequences) generated from selected database sequences (referred to as prototype regions) would detect and discriminate all serotypes of hrv and hev, a predictive model was used to assist the microarray design [ ] . this in silico model developed for predicting resequencing microarray hybridization patterns shows good concordance in the overall percentage of base calls predicted versus experimental results. thus it is possible to use this model for evaluating the performance of database sequences as potential prototype regions. in this study, we report on results of this algorithm applied to the 'utr sequences of hrv and hev and confirm that using ~ % of the rpm-flu v. / microarray ( , hrv and hev nucleotides of total , nucleotides on array) is sufficient to detect and differentiate many hrv and hev serotypes. in silico modeling figure illustrates the procedures used for the selection of hrv and hev probe sequences. first, sequences that contain the specified target region ( 'utr) and meet any selection criteria applied were downloaded from a database (currently genbank). downloaded sequences were trimmed to cover the same region using pair-wise sequence alignment. these sequences were treated as target sequences (what would be detected by the microarray) and also as prototype sequences (the potential probe sequences tiled on the microarray). each downloaded sequence was treated as a prototype used to generate probe sequences (fig . step - ) and the remaining sequences were treated as target sequences (fig . step [ ] [ ] . sets of -mer probes ( perfect match and mismatches in the th position) were generated from a prototype sequence and correspond to what would actually appear on a resequencing microarray. the other sequences were treated as a target one at a time and generated overlapping fragments from to bases long with a near neighbor Δg energy less than - . . these fragments have been shown to have strong binding strength and produce unique base calls. the generated probes and sequence fragments were the input to the in silico model [ ] for simulation which compared the fragments to the probe sets and determined the base calls a target sequence would generate. the predicted base calls were assembled into a simulated resequencing microarray result. the simulated result of a target sequence for the current prototype sequence was then run through the previously developed cibsi analysis algorithm [ ] with the following criteria. a sequence was considered detected by the cur-rent prototype sequence if at least one region of or more contiguous nucleotides was predicted to consist of a, c, g, and t base calls and no ambiguous base calls (ns). as shown in figure "yes" for "cibsi would identify" updated the list of sequences that could be detected by the current prototype sequence. this procedure was applied for every downloaded sequence. for example, if we collect sequences "a-z" from genbank, we will first use sequence "a" as a prototype sequence, then use sequences "b-z" each in turn to generate target sequences for in silico schematic of algorithm representing the prototype sequences selection process figure schematic of algorithm representing the prototype sequences selection process. a collection of database sequences covering a specified region are processed together. each sequence is treated as probe sequence that the other sequences are tested against. the numbers of these sequences detected by the probe sequences are determined. a group of sequences that are predicted to detect all the sequences is then selected. simulation. after the completion of the simulation and cibsi analysis, a list of sequences from the pool of sequences "b-z" that can be detected by prototype sequence "a" will be generated. then the cycle begins again with sequence 'b" as prototype sequence, while sequences "a, c-z" each in turn is used as target sequences to generate the list for sequence "b". the cycle will continue until we generate the list for all download sequences (sequences 'a-z"). after this is completed, the second stage of the process will be undertaken. the number of sequences that a sequence (as prototype) is predicted to detect will be sorted and ordered. the sequence that was predicted to detect the most other sequences (as targets) was selected as a probe sequence to be used in the microarray design. it was then removed from the list of sequences. all the target sequences detected by that prototype sequence were also removed from the list of sequences. this procedure was repeated until the list of sequences was empty. when two or more prototype sequences were predicted to detect the same maximal number of target sequences, one was randomly selected hence the method was non-deterministic. the process was repeated with different random seeds and the number of required probe sequences did not vary significantly while the sequences used in the microarray design could change. application: hrv and hev the described design method could be applied to any group of sequences and a minimum set of prototype regions would be determined. the group of sequences used for hrv probe design was chosen using different criteria than those used to select the hev sequences in the hev probe design due to differences in the available sequences for each in genbank. at the time of this design, only eight hrv serotypes had complete genomes sequenced. these genome sequences and all complete and partial 'utr sequences available for hrv in genbank were retrieved in april and a total of sequences were used in the predictive modeling. a set of sequences with lengths between and bp were predicted to provide detection of all those input sequences (additional file ). because hev is better characterized with complete genome sequences of all recognized serotypes, the design algorithm was applied to one complete genome sequence of each serotype. in addition, the design algorithm was applied to the d region. the design procedure generated to sequences for hev detection using 'utr region, and sequences were predicted to detect all hev d regions. it was decided to use corresponding 'utr sequences of the genomes that the d targets were selected from so that the same serotype was targeted by both target regions (additional file ). these 'utr regions were predicted to still provide complete and now redundant coverage. to assess the performance of rpm-flu v. / chip design, known hrv serotype strains obtained from atcc were tested. of the strains, had corresponding 'utr sequences tiled on the microarray and were called prototype serotypes, which were used to verify the accuracy of the designed hrv probes. the remaining strains, representing near neighbor serotypes, were selected from diverse clades based on phylogenetic classification of hrv serotypes [ , ] . these strains were used to investigate the capability of the microarray to detect other hrv serotypes that did not have their sequences tiled on the microarray. overall, the selected strains covered every single clade of hrv serotypes based on phylogenetic analysis of the p -p regions of 'utr sequences [ ] . one metric of the hybridization in a reference region is to divide the number of bases reported as a, c, g, or t by the total number of bases for that region (probe length), which we refer to as the base call rate and proportionally reflects the hybridization strength or homology between the prototype and target sequences. a hybridization profile ( fig. a) using the base call rates clearly showed a unique pattern for each serotype. the closely related serotypes with less nucleotide divergences had similar hybridization profiles across the tiled regions, so it is possible to assign species (hrva and hrvb) based only on the hybridization patterns. the brighter red spots (higher base call rates) along the diagonal suggested that stronger hybridizations between the tiled probes and 'utrs from the prototype serotypes. it is also of note that hrv does not fall into either of two major clusters which agrees with other findings that it should really be classified as a hev [ , ] . to validate the accuracy of the array clustering, 'utr of each serotype was amplified by type-specific rt-pcr and subjected to conventional sequencing. the phylogenetic tree derived from the de novo 'utr sequences (additional file ) confirms the hrva and hrvb classification. pair-wise sequence comparisons also indicated that the average nucleotide divergence of to database entries, this information could be integrated into the final identification. for these samples with high base call rates, the best hit would have tens to hundreds more matched bases than the next best hit. the remaining atcc strains represent near neighbor serotypes, which 'utr sequences share > % identifies to those of prototypes. in all but the case of hrv , the information was sufficient to identify the correct serotypes. in these samples, the difference in the number of base calls matching in the best hit and the next best hit was more variable and on average was fewer. the confidence of the identification depends on the accuracy of the base call. since the best hit always had at least one base that matched the resequencing results and was a mismatch for the next best hit, it was hybridization profiles of hrv and hev serotypes from rpm-flu v. / microarrays figure hybridization profiles of hrv and hev serotypes from rpm-flu v. / microarrays. (a) hrv serotypes were classified into two clusters corresponding to species hrva and hrvb; (b) hev serotypes (including hrv ) were classified into two clusters corresponding to heva/b and hevc/d species. base call rates (number of base calls/probe length in each tile) generated from viral samples (rows) and prototype probes (columns) were calculated and clustered using dchip software. rows standardized base call rates. positive hybridization was represented by red color. higher base call rates were shown as brighter red colors. negative hybridization (no base call) was represented by green color. the sample hrv is underlined. possible to establish the lowest confidence level that the best hit was the correct identification. the resequencing microarray's accuracy for determining the base call has been established under a variety of conditions [ ] . using this information, there is a . n that the next best hit is the most similar sequence in the database because a base is misidentified by the resequencing microarray where n is the number of mismatches between the next best hit and the matches for the best result. with . % being the largest level of uncertainty seen for these samples, it was deemed acceptable to treat the best hit as the correct identification. for hrv , several database sequences representing different serotypes had the same score and since no further information was available it was only possible to determine that a hrv species b was present. the 'utr sequences from the tested strains generated with de novo sequencing were subjected to in silico predictive modeling analysis and the result of this was used as input in the cibsi analysis program. for the case of hrv , the in silico model predicted a larger fraction of base calls being made than were observed in the experiment. for all other samples there was a good correspondence on base call fractions between model and experiment as expected. this leads us to suspect there was a processing error or sample degradation leading to the less accurate identification. a panel of hev serotypes, including serotypes from all four hev species, was similarly used to validate the specificity of rpm-flu v. / for hev detection and identification. these serotypes were originally typed based on vp sequences (personal communication -steve oberste) prototype serotypes having strong hybridization signals were designated in bold characters. the strain not identified at serotype level by microarray was underlined. *serotype identities were made by searching 'utr sequences of hrv isolates against genbank; () indicates the highest percentage of identity to the sequence in genbank. and the majority of them belonged to members of hevb. the hybridization profile ( figure b) shows distinct clusters in a similar fashion to the hrv samples based on serotypes. in this case, heva and hevb make up one cluster, while hevc and hevd (including hrv ) comprise a second cluster. this finding is consistent with the previously described clusters for hev utr sequences [ , ] . the redundancy of the targets that was a consequence of how they were selected is apparent in the more uniform response observed within each cluster to the various strains. analysis of sequences reported from rpm-flu / array analysis indicated that two levels of identifications were obtained from strains ( table ). serotype level identification was made for of the strains, in which cases correlated with typing made by the vp genes. for example, hev and coxsackievirus a (cava ), known to cause hand-foot-mouth disease, were unambiguously recognized as hev and cava respectively using rpm-flu v. / and analysis program. two strains were identified by rpm-flu v. / as hev and hev , results in agreement with the conventional sequencing of each 'utr. however, the strains as provided by cdc were identified as hev and cavb respectively, based on the vp region. specific serotypes could not be identified for the remaining sixteen samples using the sequence read generated from the array. nevertheless these samples were easily categorized into the respective species. due to amplification problems only a subset of strains were successfully de novo sequenced, which showed - % variations in 'utr sequences. the base call rates obtained by in silico predictions based on the de novo sequences were similar to the microarray results and the identifications agreed in all but one case. this study demonstrated the use of an algorithm for the design of probe sets based on an in silico predictive model [ ] , developed by our group, that minimized the probes needed for detection and identification of most serotypes of hrv and hev. the potential of using resequencing microarray for simultaneous detection and identification of highly diverse respiratory pathogens, such as hrv and hev, was also demonstrated. the conserved nature of the ' utr regions of hrv and hev genomes and the capabil- ities of the resequencing microarray allow serotype level identification of near-neighbor serotypes of hrv and hev, when long (> nucleotides) sequences are read from the array. identifications can be still made for shorter length sequences to the species level particularly when the array has one or more such sequences derived from different probes. the utility of the resequencing microarray is related to the target selection, the optimized prototype sequences represented on the array. in the case of rpm-flu v. / , the selection of hrv targets has proved to be very robust. the 'utr has been shown in this study to be a good choice for serotyping hrv on rpm, as it performed similarly well on other platforms [ , ] . all hrv variants tested in this study could be detected and identified at least to the species level. the limited number of hrv sequences available in genbank during the time of design of rpm-flu v. / rendered a few of the targets represented on rpm-flu v. / are shorter than bp. in the past year, complete genome sequences from more serotypes and another two divergent hrv'x's have been reported [ , , ] . it will be worthwhile to update the design for the next generation of the chip. in the case of hev, the rpm-flu v. / assay identified only of strains tested at serotype level. several strains not producing serotype identifications might have been indicative of assay protocol issues or probe design. the fact that the in silico model prediction was also not serotype specific indicates it was most likely a design issue. this was further confirmed by agreement in base calls made from the resequencing microarray and from conventional resequencing. although all the strains of hev have complete genome sequences, there are also many partial sequence submissions for each strain in gen-bank that were ignored for the hev design. a re-examination of the 'utr regions showed up to % difference in sequences grouped in the same serotype. this indicates that a redesign of the hev prototype regions is needed where selection of a minimal set of prototype regions would be based on all available 'utr sequence data (complete and partial) and not a subset of genome sequences. comparing the identifications made from de novo sequencing to the identifications made by cdc (sources of the samples) illustrated another shortcoming of using the 'utr region for hev that did not occur for hrv. oberste et al. demonstrated that typing based upon hev vp capsid gene sequences showed excellent correlation with serotype determined by classical antigenic methods [ ] . thus amplification and sequencing of the partial vp amino-terminal coding region has been accepted as a standard molecular typing method for hev but such is not the case for the 'utr region [ ] [ ] [ ] [ ] [ ] . our results show that the 'utr region did not correlate as closely as vp -based typing to antigenic type definitions for hev unlike how it performed for hrv. while the 'utr region is sufficient to accurately identify the groupings, a design using vp as the probe region is needed to provide serotyping identifications that will match classical methods. the current rpm design can detect and identify a more comprehensive set of viral and bacterial respiratory pathogens in parallel, including detailed discrimination of certain serotypes of hrv and hev. this study showed that most shortcomings in the design were a result of not including adequate reference sequences for the initial design. the selection of vp and d regions also showed that incorporation of primer design considerations must be contemplated sooner in the design process than it has been currently done to prevent the selection of regions that cannot be used. future development will address these limitations by reducing hev probe redundancy and lack of coverage, by updating or confirming the hrv probes to be derived from newly available hrv sequences, and by involving primer design earlier in the overall design process. a powerful feature of the expanded rpm-flu v. / resequencing pathogen microarray is that the nucleotide sequences generated from hybridization of the sample rna/dna and array-bound probe sets in conjunction with previously developed sequence analysis algorithm cibsi can be easily interpreted to make serotype or strain identifications. this feature and the platform's high resolution and high throughput aspects undoubtedly have great potential for use as a diagnostic tool, and therefore, efforts are currently underway to test the utility of this array on more clinical samples. the results presented also validated the usefulness of the design methodology and it is currently being applied to assist in a new microarray application associated with other genetically diverse viruses. a panel of cultured enterovirus (hev) prototype strains was purchased from center for disease control and prevention (cdc, atlanta, ga). the prototype strains of rhinoviruses (hrv) and hev with known titers were purchased from the american type culture collection (atcc, manassas, va). total nucleic acids were extracted from μl cultured samples by using the mas-terpure™ dna purification kit (epicentre technologies, madison, wi) and dissolved in μl of nuclease-free water. all 'utr sequences of hrv and hev with approximately bp sizes were downloaded from genbank. potential pcr primer pairs that are able to amplify - bp fragments from hrv and hev were automatically selected by a perl script primer search program developed by our group using the rules described in previous publication [ , ] . the multiplex reverse transcription polymerase chain reaction (rt-pcr) protocols for rpm-flu v. / were carried out as previously described [ ] with the following modifications. for the rt step, primer ln was replaced by primer nln (a random mer with the unique linker sequence), pg each of two internal controls nac and triosephosphate isomerase (tim), and μl of the extracted viral nucleic acids were used. the μl rt reaction product was subjected to the multiplex pcr reaction. platinum taq dna polymerase (invitrogen life technologies, carlsbad, ca) was replaced by gotaq ® dna polymerase (promega corporation, madison, wi) in the pcr reaction. primer nl instead of primer l was used with - nm each of 'utr primers in the multiplex pcr. the amplification reaction was carried out in a peltier thermal cycler -ptc dna engine tetrad (mj research inc., reno, nv) with an initial incubation at °c for min, then preliminary denaturation at °c for min followed by cycles of °c for s, - °c for s (incremental increase of °c per cycle), and °c for s, then cycles of °c for s and °c for s. microarray hybridization and processing, and the image scanning were performed according to the manufacture's recommended protocol (affymetrix inc., santa clara, ca) using a genchip resequencing assay kit (affymetrix) with modification as previously described [ ] . after scanning, gcos software was used to reduce the raw image (.dat) file to a simplified file format (.cel file) with intensities assigned to each of the corresponding probe positions. gdas software was then used to produce nucleotide reads (a, c, g and t) or base calls, comparing the respective intensities for the sense and antisense probe sets. the sequences from base calls made for each tiled region of the resequencing microarray were exported from gdas as the fasta-formatted files. base call rate refers percentage of number of base calls generated from the full length of probe in each tile. final pathogen identification for the rpm-flu v. / assay was performed using computer-implemented biological sequence identifier (cibsi) version . software [ ] , an automatic pathogen identification algorithm based on nucleic acid sequence alignment, which was developed and tested in detail in previous studies [ , ] . the ncbi blast and taxonomy databases used for cibsi analysis was downloaded in december . heat-map and clustering dendrogram was made with dchip (dna-chip analyzer, http://www.dchip.org). the rows of the imported data (base call rates) were standardized and clustered. clustering distance was -correlation with average linkage, and gene ordering by cluster tightness. 'utr sequences were amplified from hrv-or hev cdna with specific primers. amplified products were purified and sent to macrogen usa (gaithersburg, md) for automated sanger/electrophoresis-based sequencing using corresponding specific primers. phylogenetic analysis of 'utr sequences was performed by using neighbor-joining method in mega software http://www.megasoft ware.net. all nucleotide sequences used in this study are available at genbank (accession nos. eu -eu ). diagnostic system for rapid and sensitive differential detection of pathogens rapid identification and strain-typing of respiratory pathogens for epidemic surveillance applications of luminex xmap technology for rapid, high-throughput multiplexed nucleic acid detection detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray microarray-based detection and genotyping of viral pathogens pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic vibrio spp broad-spectrum respiratory tract pathogen identification using resequencing dna microarrays use of resequencing oligonucleotide microarrays for identification of streptococcus pyogenes and associated antibiotic resistance determinants using a resequencing microarray as a multiple respiratory pathogen detection assay application of broad-spectrum, sequence-based pathogen identification in an urban population identifying influenza viruses with resequencing microarrays amplicon sequencing and improved detection of human rhinovirus in respiratory samples goossens h: improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the ' noncoding regions of additional rhinovirus strains enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects a model of base-call resolution on broad-spectrum pathogen detection resequencing dna microarrays a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants genome-wide diversity and selective pressure in the human rhinovirus human rhinovirus and enterovirus represent a unique serotype with rhinovirus and enterovirus features evidence for frequent recombination within species human enterovirus b based on complete genomic sequences of all thirty-seven serotypes automated identification of multiple micro-organisms from resequencing dna microarrays highthroughput variation detection and genotyping using microarrays classification of enteroviruses based on molecular and biological properties complete genome sequences of all members of the species human enterovirus a assessing unmodified -mer oligonucleotide probe performance on glass-slide microarrays new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features molecular evolution of the human enteroviruses: correlation of serotype with vp sequence and application to picornavirus classification typing of human enteroviruses by partial sequencing of vp improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing molecular strategy for 'serotyping' of human enteroviruses molecular characterization of human enteroviruses in clinical samples: comparison between vp , vp , and rna polymerase regions using rt nested pcr assays and direct sequencing of products molecular identification and typing of enteroviruses isolated from clinical specimens the funding for this research was provided in part by the office of naval research via the nrl base program. partial support from tessarae, llc (potomac falls, va) through cooperative research and development agreement that help make this research possible is also gratefully appreciated.the opinions and assertions contained herein are those of the authors and are not to be construed as those of the u.s. navy or military service at large. zw conceived and designed the study, performed microarray experiments, analyzed data and wrote the manuscript; am designed microarray probes, analyzed data and wrote the manuscript; bl assisted in data analysis and preparing the manuscript; ck, nl and kb performed microarray experiments; dt helped to generate heatmap; ct assisted in data analyses; ds initiated the project and helped to prepare the manuscript. additional key: cord- -n tmn ph authors: cui, binglin; zhang, dangui; pan, hui; zhang, fan; farrar, jeremy; law, frieda; van doorn, h rogier; wu, beiyan; ba-thein, william title: viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: n tmn ph background: acute respiratory infections (aris) are common in children and mostly caused by viruses, but the significance of the detection of multiple viruses in aris is unclear. this study investigated respiratory viruses in aris among children and associated meteorological factors in shantou, southern china. methods: paired nasal/throat-flocked swabs collected from , children with aris, who visited outpatient walk-in clinics in a tertiary hospital between december and november , were examined for fourteen respiratory viruses - influenza viruses (flua, flub), respiratory syncytial viruses (rsv a and b), human coronaviruses (hcov: e, oc , hku , nl ), human metapneumoviruses (hmpv a and b), parainfluenza viruses (piv - ), human rhinoviruses (hrv a, b, c), enteroviruses (ev), adenoviruses (adv), human bocavirus (hbov), and human parechoviruses (hpev) - by multiplex real-time pcr. results: we identified at least one virus in . % ( / , ) and multiple viruses in . % ( / , ) of patients. ev and hrv were the most frequently detected single viruses ( . %, / and . %, / respectively) and co-detected pair ( . %, / ). overlapping seasonal trends of viruses were recorded over the year, with dual peaks for ev and single peaks for the others. by logistic regression analysis, ev was positively associated with the average temperature and humidity, hcov, and piv , but negatively with hrv, piv , and hbov. hrv was inversely associated with ev and piv . conclusions: this study reports high viral detection and co-detection rates in pediatric ari cases mainly due to ev and hrv. many viruses circulated throughout the year with similar seasonal trends in association with temperature, humidity, and wind velocity. statistically significant associations were present among the viruses. understanding the polyviral etiology and viral interactions in the cases with multiple viruses warrants further studies. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory infections (aris) are one of the illnesses of highest morbidity and mortality in children worldwide [ ] [ ] [ ] . the pathogens causing aris vary geographically and by season, but globally viruses play a major role. respiratory syncytial virus (rsv) is by far the most common pathogen associated with severe respiratory diseases as bronchiolitis, exacerbation of asthma, or pneumonia in early life, and is a leading cause of hospitalization in children under two [ ] . influenza viruses have the greatest potential to cause severe respiratory diseases in the very young, the elderly and those with underlying chronic conditions [ ] . enteroviruses including human rhinoviruses (hrv) and human enteroviruses (ev), previously identified in childhood upper respiratory tract infections, are commonly associated with milder aris and have been suspected as major etiological agents of lower respiratory tract infections leading to bronchiolitis and pneumonia in infants [ ] . it has also been reported that human metapneumovirus (hmpv) causes approximately - % of all aris in children and adults [ ] and adenoviruses (adv) account for - % of respiratory infections in children [ ] . respiratory illnesses can be attributable to other viruses such as parainfluenza viruses (piv) and human coronaviruses hcov- e, oc [ ] . with rapid progress in molecular diagnostics, newly discovered viruses including human bocavirus (hbov), human coronaviruses (hcov-nl , hcov-hku ), human parechoviruses (hpev), and polyomaviruses wu (wupyv) and ki (kipyv) have also been detected in children with respiratory infections, with varying levels of proof of causation [ ] . hospital-based studies in children published over the last decade worldwide have identified viruses in up to % of ari episodes, with a single virus found in - % and multiple viruses in - % of infected patients [ , , ] . coinfection is reportedly related to the time of year when circulations of multiple viruses occur [ ] . some studies have shown that the prevalence of co-infections is not related to the absolute prevalence of individual viruses [ ] . factors such as young age, male gender, and history of immunosuppression are associated with an increased chance of viral co-infections [ , , ] . there could be likely interactions between climatic, environmental, and behavioral factors, and complex interplay between circulating viruses and population-level immunity regarding viral coinfections. understanding these factors may help us prevent transmission of these infections. recent etiologic studies on pediatric respiratory infections mostly report the prevalence in hospitalized children and the seasonality of viruses without elaborating viral co-infection. therefore, the significance of the detection of multiple viral pathogens in aris is unclear. here, we investigated fourteen common respiratory viruses among pediatric outpatients in southern china during - and their associations with meteorological factors. this study was conducted at the pediatric outpatient walk-in clinics, the first affiliated hospital of shantou university medical college. the pediatric department provides both primary and tertiary care (common practice in china) for approximately , children per year in the chaoshan region of southern china. the chaoshan region is in the subtropical zone with an average annual temperature of . °c and excellent to lightly polluted air quality levels (air quality index, aqi: - , in - ) . based on modified who standard case definition of aris [ ] , eligible participants were defined as a child - years of age presenting within days of onset of illness with at least two of the following: fever, sore throat, cough, rhinorrhea, nasal congestion, and hoarseness of voice. patients with any condition preventing swab collection were excluded. we recruited eligible patients in the morning, during which approximately % of patient visits are made, on a daily basis except public holidays from december to november . participants' demographic details and clinical features are shown in table . paired nose and throat-flocked swabs (copan, brescia, italy, cat. no. cs and cs ) were collected from each participant, combined in one tube, and stored within h of collection at − °c until further processing. multiplex real-time pcr was performed using roche, lightcycler ii (roche diagnostics, penzberg, germany) to identify the following respiratory viruses: influenza a (flua), influenza b (flub), respiratory syncytial viruses a and b (rsv), human coronaviruses e, oc , hku and nl (hcov), human metapneumoviruses a and b (hmpv), human parainfluenza virus types , , , and (piv , piv , piv , and piv ), human rhinoviruses a, b, and c (hrv), human enteroviruses (ev), human adenoviruses (adv), human bocavirus (hbov), and human parechoviruses (hpev). nucleic acid extraction was performed using the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany, cat. no. ). reverse transcription and realtime pcr assays were performed as described previously [ ] , except for the primers and/or probes for hrv, hpev, and internal control equine arteritis virus (eav, see the sequences of viruses in additional file ). due to known cross-reactivity between enteroviruses [ ] [ ] [ ] , hrv was detected using two sets of primers and probes: hrv-v (version ) for screening and hrv-v (version ) for confirmation. real-time pcr results were interpreted as described previously [ ] . the pcr was considered positive or negative when the cp value was less than cycles or exceeded cycles, respectively, and the positive control showed the expected cp value, negative control was negative, and internal control showed the expected cp value. a negative internal control signal was accepted in case of a positive target sequence with correct positive and negative control signals. meteorological data, including the average daily temperature (°c), the average daily humidity (%), and the average daily wind velocity (km/h), were collected from the official website of shantou meteorology, tutiempo. net (http://www.tutiempo.net/en/climate/shantou/ / .htm). we used chi-square test to compare differences in the distribution of categorical variables, anova and kruskall wallis tests to compare medians, and the pearson correlation analysis to evaluate the associations between the meteorological factors and viruses and among viruses. the variables with significant associations were further analyzed in multivariate logistic regression models, in which symptoms and positivity of viruses were treated as dependent and independent variables to assess virussymptom associations; and individual viruses were treated as dependent variables with meteorological factors or other viruses as independent variables to investigate meteorological factor-virus and virus-virus associations. a two-tailed p-value of < . was considered significant. all these analyses were performed with spss statistics version . . the study was approved by the ethics committee of the first affiliated hospital of shantou university medical college and the oxford university tropical research ethical committee (oxtrec). written informed consent was obtained from parents or legal guardians of children enrolled in the study. of , children ( . % male) recruited, . % ( / , ) were > - years old (table ). at least one virus was identified in . % ( / , ) of the patients, with single virus in . % ( / , ) and multiple viruses in . % ( / , ). hpev was not detected. compared with virusnegative patients, virus-positive patients were less likely to have fever (or: . , % ci: . - . , p = . ). patients with multiple viruses were more likely to have rhinorrhea than those with single virus (or: . , % ci: . - . , p < . , table ). hcov (or: . , % ci: . - . ) and piv (or: . , % ci: . - . ) were more prevalent in the > year age group than in the ≤ year group (all p ≤ . ), while hbov (or: . , % ci: . - . ) and rsv (or: . , % ci: . - . ) were less frequently found in the > year group (all p < . ). chi-square test and multivariate logistic regression analysis showed that cough was positively associated with hrv and rsv, and negatively with ev; rhinorrhea was positively associated with hrv, piv , and hbov, and negatively with ev; fever was positively associated with ev, and negatively with hrv and piv ; and nasal congestion was positively associated with rsv, and negatively with ev and hcov (all p < . , table ). viruses detected alone or co-detected with other viruses are shown in table . the most frequently detected virus was ev ( . %, / ), followed by hrv ( . %, / ), and hcov ( . %, / ). ev and hrv were most commonly co-detected with other viruses (table ) and also the most commonly co-detected pair of viruses ( . %, / , see the distribution pattern of viruses in additional file ). screening with hrv-v identified cases co-positive for hrv and ev, and subsequent confirmation with hrv-v primers/ probes [ ] resulted in only positive cases ( . %, / ). the temporal circulation and co-circulation patterns of viruses are shown in figures and . there were overlapping seasonal trends of many viruses throughout the year, with dual peaks for ev in july and september and single peaks for the other viruses. both ev and hrv circulated throughout the year. hcov and piv circulated predominantly between april and may but sporadically throughout the year. piv , rsv, flua, and adv peaked in january, while hbov peaked in march. flub circulated mostly from february to july with a peak in april. codetection of - viruses occurred all in may (see additional file ). the optimal average daily temperature, humidity, and wind velocity for these viruses are shown in table . table shows the multivariate logistic regression models for independent associations between the viruses and meteorological factors and between the viruses. ev was positively associated with the average temperature and humidity and the presence of hcov and piv , but negatively with hrv, piv , and hbov. hrv was negatively associated with the presence of ev and piv . hcov was positively associated with the average temperature and humidity and the presence of ev and piv . piv was positively associated with the average humidity and the presence of rsv and flua, but negatively with the average temperature and wind velocity, and the presence of ev, hrv, and hbov. piv was positively associated with the average temperature and the presence of hcov and rsv, however, negatively with the wind velocity. hbov was positively associated with rsv and flua, but negatively with the average temperature and humidity and the presence of ev and piv . rsv was positively associated with the presence of piv - , hbov, and flua, but negatively with the average temperature and wind velocity. flua was positively associated with the presence of piv - , hbov, and rsv, but negatively with the average temperature. this is the first prospective study reporting the associations between meteorological parameters and co-circulation patterns of common respiratory viruses. the viral detection rate among pediatric outpatients with aris in this study ( . %, / , ) was higher than those reported from nanjing, china ( viruses, . %, / ) [ ] and other countries, including honduras ( viruses, . %, / ) [ ] and greece ( viruses, . %, / ) [ ] in the same study period. enteroviruses (ev, . % and hrv, . %) were most frequently detected in our outpatient children. influenza viruses and rsv, the leading pathogens in pediatric outpatients in only statistically significant results (p < . by chi-square test for individual comparisons of proportions within each group) are shown as "√" with reference(s) shown as "-". the optimal temperature for hcov and hbov was - °c. the optimal relative humidity was - % for piv and - % for hcov. similar studies [ , [ ] [ ] [ ] , were detected in . % and . % of our cases, with hcovs ( e, oc , hku , and nl ) in . %, and relatively recently discovered viral pathogens hbov and hmpv in . % and . % of cases, respectively ( table ) . the viral co-detection rate ( . %, / , ) was also high among our study population. reported rates of codetection vary widely, from . % among pediatric patients with influenza-like illness [ ] to % among infants with acute bronchiolitis [ ] . detection of dual viruses is common, and co-detection of five [ ] or even six viruses [ ] is not anecdotal. all the cases with - viruses in this study were in may, the end of the cold season in the chaoshan region. this may be in part due to past viral infections, as some viruses can still be detectable by pcr several weeks after infection [ , ] . most studies have shown that rsv is the predominant respiratory pathogen co-detected in hospitalized children, followed by hrv, piv, hmpv, hbov, and flua [ , ] . in this study, ev, hrv, hcov, and piv - were involved in the majority of co-detections, with ev-hrv as the most frequently co-detected pair ( % of codetections). ev and hrv were included in the panels in many studies globally [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and the ev-hrv pair was the most commonly detected pair among outpatient children with aris in finland ( . % of codetections) [ ] and infants with acute bronchiolitis in brazil [ ] . the co-detection rate of ev-hrv in this study is similar to that in finland [ ] . varying detection rates of multiple viruses in different studies may reflect the differences in the study period and location, study population, environmental factors, the number of respiratory pathogens tested, and/or the diagnostic methods/techniques used. likely reasons behind high detection rates of single and multiple viruses in this study could be due to improved recovery of viruses by using flocked swabs [ ] and/or combined nasal and throat swabs [ ] . there are advantages and disadvantages of multiplex pcr technique in diagnosing respiratory viral infections. while its high sensitivity and specificity facilitate simultaneous detection of a large spectrum of viruses, including those difficult to be identified by traditional methods [ ] , its capacity to detect low amounts of viral nucleic acids in some cases during viral incubation period, asymptomatic infection, or post-infectious shedding makes it difficult to interpret the results [ , ] . the development and validation of standardized quantitative pcr with clinically relevant cutoff values [ ] or combining qpcr with serology could be helpful for etiologic understanding of simultaneous presence of multiple viruses. certain host-specific risk factors may predispose a child to respiratory co-infection. younger age [ , , ] , male gender, and history of immunosuppression are associated with increased risk of viral co-detections [ ] . nonetheless, similar associations were not found in this study. viral co-detection is not random; clear associations for certain viral co-occurrence have been described [ ] . the viruses circulating at the same time of a year are more likely to accompany each other [ , , ] . this may be driven by meteorological factors which actually work behind seasonal variations, or by interactions of certain coexisting viruses. temperature, humidity, and wind velocity are the most commonly studied factors significantly associated with the overall number of ari hospitalizations and the prevalence of various respiratory viruses [ ] [ ] [ ] . the average temperature is the key climatic parameter associated with the prevalence of many respiratory viruses. some viruses survive and/or replicate better at low temperatures, having peak prevalence in the colder months. in our study, the detection rates of piv , rsv, flua, and adv were negatively associated with temperature and were highest at temperatures between °c and °c (tables and ), supporting the notion that low temperature is suitable for the survival of lipidenveloped air-borne viruses [ ] . low temperatures have been found to favor rsv in southeast china [ ] , malaysia [ ] , nepal [ ] , brazil [ ] and germany [ ] , influenza in japan [ ] and germany, and adv in germany [ ] ; however, high temperatures favored piv in southeast china [ , ] and nepal [ ] , rsv in singapore [ ] , hong kong [ ] , and indonesia [ ] , and adv in southeast china [ ] . no association between temperature and flua activity was found in nepal [ ] and brazil [ ] . in our study, other viruses such as ev, hcov, and piv were more often detected during months with higher temperatures, having peaks at temperatures between °c and °c (tables and ). in contrast to our findings, hcov was negatively associated with temperature, and no association between ev and temperature was found among children with aris in germany [ ] . association of humidity and viral detection rates has been reported from germany [ ] , singapore, hong kong, brisbane, and vancouver [ ] . in this study, three viruses (ev, hcov, and piv ) were positively associated with the average humidity ( table ). the optimal average humidity ranges for ev and hcov were - % and - % respectively, supporting a previous finding that high average humidity ( %) had a protective effect on the survival of hcov [ ] . our findings on piv are inconsistent with animal and laboratory observations that lipid-enveloped viruses such as piv survived better in cooler, less humid environment [ ] . hbov was negatively associated with the average humidity, and its optimal humidity was - % (tables and ). no climatic data is available for comparison regarding this virus. previously reported association between flua and the average humidity [ ] was not found in our study. wind velocity piv and piv have been reported to be negatively associated with wind velocity [ ] . in low wind speed environment, viruses can easily colonize in the epithelium of upper respiratory tract [ ] . an increased wind velocity is correlated with rsv activity in germany [ ] . in our study, piv , piv , and rsv were inversely associated with the wind velocity. although we observed higher rates of ev and flub but lower rate of hrv in low wind velocity, we could not confirm these associations by logistic regression analysis. the underlying reasons for the observed associations between virus circulations and meteorological factors are unclear. climate could have a direct or indirect effect on viral survival, transmission efficiency, host immunity, and social behavior change [ , ] . cold and dry conditions might favor the transmission of viruses, and cold or rainy days could decrease outdoor activities of children and increase the probabilities of close contact and transmission of infections [ ] . holidays (supported by our data with less cases in february as chinese new year and july-august summer holiday, figure and additional file ), could also play a role in an annual epidemic cycle [ ] . it is likely that several factors interact in complex ways in the development of observed epidemics under optimal climatic conditions and that the contributions of individual factors vary for different viruses. further investigations such as time series model over many years are needed to account for their inherent autocorrelations [ ] , and thus the observed associations between meteorological parameters and viruses in this exploratory analysis should be interpreted with caution. viral co-detection patterns may be the reflection of interactions between viruses. co-detection of viruses has been frequently reported [ , , , , , ] . here, we have assured their associations by mathematical models (table ) . we identified many pairs of viruses with positive associations, including ev-hcov, hcov-piv , piv -rsv, piv -flua, piv -rsv, hbov-rsv, hbov-flua, and rsv-flua. negative associations for ev-hrv, ev-piv , ev-hbov, hrv-piv , and piv -hbov were also found in this study. both belonging to the enteroviruses genus, hrv and ev have similarities in the highly conserved sequence of the ' noncoding region, which is the preferred site for molecular assay development [ , , , ] . cross-reactivity between the primers of hrv with evs has been reported and is among others attributable to ev-d , an emerging pathogen frequently undetected and misdiagnosed as hrv [ , , , ] . confirmation of cross-reacting ev types in this geographic region should be done in future studies. there is no consensus in the literature on the clinical implications of the viral detection and co-detection. some studies linked multiple viral detections with fever [ ] , or increased hospitalization and intensive care admission [ ] , while others described a very similar prognosis as in single infection [ , , ] , or even milder presentations [ ] . in this study, the virus-negative patients had fever more often, which may be caused by other pathogens such as bacteria. we also found that rhinorrhea was more frequently present in patients with multiple viruses than in those with a single virus, and some viruses were more (or less) likely to exist in certain age groups or were accompanied with certain symptoms. since we did not follow the cases, the associated clinical course and outcome (such as hospitalization) remain unknown. a better understanding on the clinical courses of single and multiple viral etiologies requires further studies. the current study has several limitations. the majority of outpatients enrolled in this study were mild and moderate cases. therefore, we could have missed pathogens responsible for severe aris. as healthy or asymptomatic controls were not included, their viral carriage burdens and the actual role of virus infections could not be elucidated. following up the cases for clinical burdens and serologic testing would be required in future studies. air quality indicators such as ozone and pm . , which might influence the host's susceptibility or virus circulation, should be included to investigate meteorological factors. in summary, this study reports a high viral carriage in pediatric ari cases with high viral co-detection rates mainly due to ev and hrv. there were overlapping seasonal trends of many viruses throughout the year. meteorological factors, including temperature, humidity, and wind velocity, were associated with the viral detection rates. statistically significant associations were present among the viruses. further studies are needed to address polyviral etiology and viral interaction in multiple virus positive cases. additional file : primers and probes of viruses. additional file : the distribution pattern of viruses in pediatric outpatients with acute respiratory infections, aris (n= ). the authors declare that they have no competing interests. authors' contributions blc and hp designed and performed the experiments, analyzed the data, and wrote the paper. dgz designed and performed the experiments, and analyzed the data. fz analyzed the data. jf conceived and designed the experiments, and analyzed the data. fl analyzed the data, and facilitated the study. hrvd analyzed the data. byw facilitated the study. wb-t designed the experiments, analyzed the data, and wrote the paper. all authors read and approved the final manuscript. temperature, nitrogen dioxide, circulating respiratory viruses and acute upper respiratory infections among children in taipei, taiwan: a population-based study infection and disease in a group of south indian families. i. introduction, methods, definitions and general observations in a continuing study acute respiratory illness in an american community. the tecumseh study the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis evaluation of viral co-infections in hospitalized and non-hospitalized children with respiratory infections using microarrays viral etiology of influenza-like illnesses in antananarivo human adenovirus infection in children with acute respiratory tract disease in guangzhou the role of infections and coinfections with newly identified and emerging respiratory viruses in children high rate of viral identification and coinfections in infants with acute bronchiolitis viruses in community-acquired pneumonia in children aged less than years old: high rate of viral coinfection impact of viral infections in children with community-acquired pneumonia: results of a study of respiratory viruses multipathogen infections in hospitalized children with acute respiratory infections polymicrobial acute respiratory infections in a hospital-based pediatric population development and evaluation of a four-tube real time multiplex pcr assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses clusters of acute respiratory illness associated with human enterovirus -asia, europe, and united states multi-centre study in the netherlands examining laboratory ability to detect enterovirus , an emerging respiratory pathogen surveillance of respiratory viruses in patients with influenza-like illness in nanjing etiology and seasonality of viral respiratory infections in rural honduran children acute viral respiratory infection in children under years: epidemiological study in two centers in respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from rna viruses in community-acquired childhood pneumonia in semi-urban nepal; a cross-sectional study broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system co-infections with influenza and other respiratory viruses the epidemiology and etiology of influenza-like illness in chinese children from to mixed respiratory virus infections viral etiology of common cold in children associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy multiple simultaneous viral infections in infants with acute respiratory tract infections in spain viral etiology of influenza-like illnesses in cameroon viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections comparison of nasopharyngeal flocked swabs and nasopharyngeal wash collection methods for respiratory virus detection in hospitalized children using real-time polymerase chain reaction evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections are meteorological parameters associated with acute respiratory tract infections? characteristics and the prevalence of respiratory viruses and the correlation with climatic factors of hospitalized children in suzhou children's hospital seasonal influenza activity in hong kong and its association with meteorological variations comparison of the incidence of influenza in relation to climate factors during - in five countries epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of years seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region a seasonal model to simulate influenza oscillation in tokyo association of meteorological factors with childhood viral acute respiratory infections in subtropical china: an analysis over years seasonal trends of viral respiratory tract infections in the tropics epidemiology of respiratory syncytial virus infection among paediatric patients in hong kong: seasonality and disease impact climatic, temporal, and geographic characteristics of respiratory syncytial virus disease in a tropical island population effects of air temperature and relative humidity on coronavirus survival on surfaces incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong distribution and risk factors of pandemic influenza a (h n ) in mainland china comparison of the nuclisens basic kit (nucleic acid sequence-based amplification) and the argene biosoft enterovirus consensus reverse transcription-pcr assays for rapid detection of enterovirus rna in clinical specimens continued seasonal circulation of enterovirus d in the netherlands rhinovirus c and respiratory exacerbations in children with cystic fibrosis submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank the pediatricians from the pediatric department, the first affiliated hospital of shantou university medical college for their generous support, the children and their guardians for participation in this study, richard molenkamp at the university of amsterdam, academic medical center for technique and knowledge transfer to set up the multiplex real-time pcr, jieling chen at the shantou-oxford clinical research unit for technical assistance, and staff in the international institute of infection and immunity, shantou university medical college for their assistance with real-time pcr. this study was supported by the li ka shing foundation, shantou university medical college, and the university of oxford (grant no. b rsrt - ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord- -mean sj authors: giamberardin, heloisa i.g.; homsani, sheila; bricks, lucia f.; pacheco, ana p.o.; guedes, matilde; debur, maria c.; raboni, sonia m. title: clinical and epidemiological features of respiratory virus infections in preschool children over two consecutive influenza seasons in southern brazil date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: mean sj this study reports the results of a systematic screening for respiratory viruses in pediatric outpatients from an emergency department (ed) in southern brazil during two consecutive influenza seasons. children eligible for enrollment in this study were aged – months and presented with acute respiratory symptoms and fever. naso‐ and oropharyngeal swabs were collected and multiplex reverse transcription pcr (rt‐pcr) was performed to identify the respiratory viruses involved. in total, children were included in this study: in and in . in , samples ( %) were found to be positive for at least one virus and the most frequently detected viruses were human rhinovirus (hrv) ( %), adenovirus (adv) ( %), and human coronavirus (cov) ( %). in , samples ( %) were found to be positive for at least one virus, and the most frequently detected were hrv ( %), adv ( %), and enterovirus ( %). further, the presence of asthma (or, . ; % ci, . – . ) was independently associated with hrv infection, whereas fever was associated with adv (or, . ; % ci, . – . ) and influenza infections (or, . ; % ci, . – . ). ten patients ( %) were diagnosed with pneumonia, and six of these tested positive for viral infection ( hrv, rsv, and adv). thus, this study identified the most common respiratory viruses found in preschool children in the study region and demonstrated their high frequency, highlighting the need for improved data collection, and case management in order to stimulate preventive measures against these infections. j. med. virol. : – , . © wiley periodicals, inc. viral acute respiratory infections (aris) in pediatric outpatients represent a significant burden on emergency departments (eds) and the patients' families, mainly during influenza seasons, being associated with around % of all deaths in pre-school children worldwide, with % of these deaths due to pneumonia. [izurieta et al., ; fiore et al., ; bezerra et al., ; gessner et al., ; munywoki et al., ] . however, information about the etiology of viral aris in preschool children, particularly in outpatients, is limited, especially in latin america. community respiratory viruses (crvs) are frequently found in this population and could be involved in severe infections in young children and patients with comorbid conditions [ampofo et al., ; ampofo et al., ; bender et al., ] . therefore, understanding the dynamics of their circulation among various age groups is essential, not only to help pediatricians in their diagnosis but also for developing preventive measures against these infections. curitiba city in southern brazil has a temperate climate with low temperatures and high humidity in winter. previous studies have shown a seasonal pattern of prevalence of respiratory viruses in curitiba, with an increased incidence in winter. during this period, influenza and respiratory syncytial viruses are most frequently associated with increased respiratory disease morbidity and lead to an extensive respiratory disease burden [tsuchiya et al., ; coelho et al, ; debur et al., ; raboni et al., ] . however, the majority of these studies have been conducted in hospitalized patients or outpatients screened under an influenza surveillance program. although reports that a high number of outpatients consistently present with aris, few of them have evaluated the pathogens involved in aris in this group of patients [pavia, ; adams et al., ] . this study reports, the results of a laboratory-based surveillance for respiratory viruses in preschool children who were treated in the ed of a pediatric referral hospital during two consecutive influenza seasons. a cross-sectional study was carried out from july to november in both and . children considered eligible for this study were outpatients who were aged - months and attended the ed of pequeno pr ıncipe hospital (pph), curitiba, brazil; these children had been diagnosed with acute upper or lower respiratory infections or asthma-related conditions. patients whose parents signed the consent form, completed the form (demographic, clinical, and influenza vaccine data), and met all of the following inclusion criteria were enrolled in the study: (i) aris with or without fever (> ˚f/ ˚c); (ii) that occurred < days before the ed visit; and (iii) for which samples had been obtained. each patient was included only once. patients exhibiting symptoms that began > days before the ed visit, patients who were administered oseltamivir during the days ( doses) prior to enrollment, and those from whom samples could not be obtained were excluded from the study. detailed demographic information was obtained for all children enrolled by using medical charts and brief interviews. in addition, clinical data, outcomes, comorbidities, and the time spent in school or a day-care center were assessed for each child. aris comprise a large and heterogeneous group of diseases such as the following: flu syndrome; common cold; pharyngitis; laryngitis; tracheitis; asthma and wheezing crisis; bronchiolitis; and pneumonia of bacterial, viral, and other etiologies. this study was approved by the institutional review board (irb: # - ), and written informed consent was obtained from the individuals (parents, tutor, or relatives) responsible for the patients. the pequeno pr ıncipe hospital is the largest pediatric referral hospital of curitiba and attends children referred from various health units in the city and metropolitan region for tertiary and quaternary medical care. it also has a large number of specialized clinics and emergency rooms for primary and secondary medical care, the study was conducted with these group of patients. samples from naso-and oropharyngeal mucosa were collected with swabs and transferred to virological transport media (tryptose phosphate buffer enriched with gelatin) and shipped at ˚c to the virology laboratory. crvs were detected using multiplex rt-pcr. the viral genome was extracted using the viral gene-spin tm kit (intron biotechnology inc., korea) according to the manufacturer's instructions. the multiplex rt-pcr technology used for the samples from enables simultaneous detection of viruses (seeplex rv ace detection, seegene, korea): human adenovirus (adv); human coronavirus (cov) types e/nl and oc /hku ; human metapneumovirus (mpv); parainfluenza virus types , , and (piv- , piv- , and piv- ); influenza a (flua) and influenza b (flub) viruses; respiratory syncytial virus types a and b (rsv-a, rsv-b); and human rhinovirus types a and b (hrv a/b). the multiplex rt-pcr technology used for the samples enables simultaneous detection of viruses (seeplex rv ace detection, seegene, korea): the viruses of the seeplex rv ace kit plus human enterovirus (ev), human bocavirus (bov), and parainfluenza virus type (piv- ). subtyping of influenza a viruses was performed by using real time rt-pcr (qrt-pcr) according to the cdc protocol [who, ] . sample size estimation was based on a % bilateral confidence interval for the expected proportion of children with infection caused by influenza virus. the expected proportion was assumed to range from . to . , the number of subjects included was , and the maximum confidence interval precision (range) was chosen to be . . data were compiled using jmp version . . (sas institute inc., cary, nc) and analyzed using the r package version . . (r core team; ). descriptive statistics were used to describe the general characteristics of the study population. quantitative variables with normal and non-normal distributions are presented as means ae standard deviation and medians with interquartile ranges (iqrs, th- th percentiles), respectively, whereas qualitative variables have been expressed as numbers and percentages with % confidence intervals (cis). chi-square or fisher's test were used for comparison of qualitative variables. the student's t-test or the mann-whitney u-test was applied to compare quantitative variables, as appropriate. five viruses (hrv, adv, piv, flu, and cov) were selected for a binomial analysis to assess the impact of viruses on the health of the children. the presence or absence of the virus was compared with different variables (demographic, clinical features, and host characteristics). the selection of these viruses was based on either the high frequency of infections or previously reported severity of infections caused by them. only cases with mono-detection were included in this evaluation. variables with an associated p-value < . in the univariate analysis were subjected to multivariate logistic regression to identify independent predictors of these viruses infection. odds ratios (ors) and % cis were calculated. all p-values were based on two-tailed comparisons, and the level of significance was set at p < . . initially, patients were enrolled, of these were later excluded because: (i) inappropriate sample collection ( ); (ii) date of onset of symptoms ! days ( ); (iii) medical record was not found ( ); (iv) not meeting ili acute condition ( ); and (v) age > months ( ). a total of children between the ages of and months were included in the study during two influenza seasons ( in and in ). the mean age of the children was . (ae . ) months, and ( %) were male individuals. further, ( %) of the children attended school or daycare centers, of which ( %) attended school or day care on a fulltime basis. comorbid conditions were present in ( %) children, with asthma/bronchitis (n ¼ , %, as evidenced by reporting of previous episodes of wheezing with the use of bronchodilator medication) being most frequently reported (table i) . fever, cough, and pharyngeal erythema were the most common clinical manifestations. chest radiography was performed for ( %) patients, and perihilar infiltrates, and pulmonary consolidation were found in / ( %) and / ( %) cases, respectively. six of the patients ( %) with pulmonary consolidation tested positive for multiple viruses ( hrv, rsv, and adv). nine ( %) patients required non-invasive respiratory support, and antibiotics were prescribed for ( %) children. nasopharyngitis and influenza-like infection were diagnosed in % of the cases (table ii) . clinical complications occurred in eight ( . %) patients: one had sinusitis, two presented with otitis media, one had tracheobronchitis, and one had pneumonia. of the children evaluated in , ( %) samples were positive for at least one respiratory virus. hrv a/b (n ¼ , %), adv (n ¼ , %), and cov /nl (n ¼ , %) were detected most frequently. viral co-infections occurred in ( %) patients, mainly with the association of hrv a/b and ( ) adv (n ¼ ; %). in , out of ( %) samples were positive for at least one respiratory virus. further, hrv a/b (n ¼ , %) and adv (n ¼ , %) were detected most frequently, followed by enterovirus (n ¼ , %). viral co-infections occurred in ( %) patients and mainly involved an association of hrv a/b and adv (n ¼ , %), enterovirus and hrv a/b (n ¼ , %), or hrv a/b and parainfluenza (n ¼ , %) (table iii, fig. ). the distribution of the frequency of the viruses according to the age group showed that most of them were detected in children aged between and months, except for influenza a and human coronaviruses. notably, in both years, there was a higher rate of detection of influenza type b ( %; / ) than of type a ( . %; / ). while of the samples containing influenza a were of subtype h n , one was of an undetermined subtype ( . %), and none of them belonged to subtype h n pdm . there was a fluctuation in the most frequently detected respiratory viruses according to the epidemiological week during the study, with a higher frequency of detection of influenza b in the first weeks of the study, followed by influenza a h n . the proportion of influenza virus infection in the population studied was between % and % ( % ci, . - . ). further, a significant difference in the coverage rate of influenza vaccination was observed between and ( %; % ci, . - . vs. %; % ci, . - ). over % of immunized patients received the monovalent h n pdm influenza vaccine in , whereas in , most of the immunized children received the trivalent vaccine (table iv) . binomial analysis of the five selected viruses showed that the presence of underlying asthma/bronchospasm was significantly associated with hrv infection (or, . ; % ci, . - . ), while fever and myalgia was less likely to be associated with hrv. on the other hand, the presence of fever was significantly associated with adv and influenza infections (or, . ; % ci, . - . and or, . ; % ci, . - . , respectively), whereas the presence of underlying conditions did not increase the chance of acquiring these infections. none of the evaluated variables showed significant correlation with the presence of parainfluenza virus and coronavirus infections (table v) . this study showed crv circulation in a population of preschool children in post-pandemic years. human rhinovirus and adenovirus were the main viruses found in the years following the pandemic. the profile of detected viruses was different from that previous reported, and viruses such as rsv were less frequent, while adenoviruses, which are generally associated with severe infections in hospitalized patients, were more frequently detected. this is probably due to differences in age groups, because previous studies with pediatric outpatients under years old showed a pathogen profile similar to that of hospitalized children [milstone et al., ; hara et al, ; adams et al., ] . s cis cek et al. [ ] studied respiratory viruses in adult and pediatric patients in a -year follow-up study. similar to our results, in pediatric patients, they found that hrv was detected more frequently in outpatients, and rsv, in inpatients; however, the distribution of the detected viruses was not stratified according to age group. moreover, diagnosis was based on antigen detection and virus isolation, which are less sensitive than the method used in this study. likewise, zimmerman et al. [ ] re-] reported a higher frequency of rsv and influenza a viruses in outpatients, although they were between months and years of age, unlike the group assessed in this study. when comparing the two periods studied, we observed a higher prevalence of viral coinfection in , due to increased virus detection owing to the use of rv multiplex pcr. however, in both years, the coinfection rates were lower than those in various previous reports, wherein they ranged from % to % [lepiller et al., ; luchsinger et al., ; turunen et al., ] . though, most of these studies were carried out in hospitalized children who generally are younger and presented with their first episodes of viral respiratory infection. the frequency of infection caused by coronaviruses, which are usually associated with common cold, was . %, a rate higher than that previously reported [pilger et al., ; hara and takao, ] . the molecular method used did not allow genotype differentiation, but in general, patients infected with coronavirus showed favorable outcomes, without reports of any complications. several factors contributed to the low influenza a h n pdm circulation in and . first, vaccination coverage rates were high owing to the public immunization campaigns in and : vaccination coverage in the studied age group in the city of curitiba was almost % [brazilian health ministry, ] . in addition, because of the magnitude of the pandemic in southern brazil, the population can be considered sensitized during the study period. second, the early use of oseltamivir might be a contributing factor. third, a high rate of influenza a h n pdm infection was observed in [libster et al., ] , which might have led to a protective effect in many children during the subsequent season. no gender-specific differences were found in the prevalence of crv infections, which were generally of low severity. the incidence of crvs was higher in children attending school and/or day-care centers; however, there was no significant difference in the amount of time spent in these places in the years. similar to previous reports, in both seasons, the most prevalent underlying conditions associated with crv infection were asthma and wheezing crisis [tregoning and schwarze, ; turunen et al., ] . x-rays were performed in only % of the patients, and perihilar infiltrates were the most common radiologic findings, although none of the patients needed mechanical ventilation or intensive care treatment. one of the main challenges in the care of patients with aris is to determine clinical and demographic factors that can help in the etiological diagnosis of this infection. for this reason, we selected five rvs that are often detected in aris and used logistic regression to evaluate correlations between their prevalence and various parameters. an association was observed between hrv and asthma, and between the presence of fever and hadv and influenza infections. similarly, previous reports have linked hrv to atopic characteristics, and the susceptibility to hrv-induced wheezing has been recognized as an important risk factor for childhood asthma [tregoning and schwarze, ; mackay et al., ; turunen et al., ] . the strongest association was observed between fever and adenovirus and influenza infections, suggesting that screening for influenza by rapid tests would be recommended. as previously reported, children maintain the chain of transmission of influenza since they shed viruses for a more prolonged time and in higher concentrations; thus, control of these infections must be based on disease control in pediatric patients [ploin et al., ; d'onise and raupach, ; fiore et al., ] . this study had some limitations: (i) the difference in the number of viruses detected by the multiplex rt-pcr kits used in the years may have influenced the detected virus profile, although no significant difference was found in the positivity rates; and (ii) the predefined sampling period, made it impossible to assess the seasonality of the infections, although this information is already available for the studied region. despite the advances in the establishment of a wide network of information on the circulation of influenza viruses, little is known about vaccination coverage as well as the impact of crvs in preschool children. our findings provide novel insights into the epidemiology and clinical impact of respiratory viruses on pediatric health and highlight the need for implementation of surveillance programs for respiratory viral infections, seeking to stimulate vaccination and to guide preventive measures to protect this population. bold values, statistical differences. comparison of human metapneumovirus, respiratory syncytial virus and rhinovirus respiratory tract infections in young children admitted to hospital seasonal invasive pneumococcal disease in children: role of preceding respiratory viral infection epidemiology, complications, and cost of hospitalization in children with laboratory-confirmed influenza infection development and validation of a risk score for predicting hospitalization in children with influenza virus infection viral and atypical bacterial detection in acute respiratory infection in children under five years si-pni sistema de informas cão do programa nacional de imunizas cão impact of respiratory infections by influenza viruses a and b in pediatric patients from federal university of parana, brazil the burden of influenza in healthy children in south australia impact of human metapneumovirus infection on in and outpatients for the years - in southern brazil centers for disease control and prevention. prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) seasonal influenza epidemiology in sub-saharan africa: a systematic review three-year study of viral etiology and features of febrile respiratory tract infections in japanese pediatric outpatients coronavirus infections in pediatric outpatients with febrile respiratory tract infections in hiroshima, japan, over a -year period influenza and the rates of hospitalization for respiratory disease among infants and young children high incidence but low burden of coronaviruses and preferential associations between respiratory viruses pediatric hospitalizations due to influenza in in argentina comparison of virological profiles of respiratory syncytial virus and rhinovirus in acute lower tract respiratory infections in very young chilean infants, according to their clinical outcome community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a -month period epidemiology of respiratory viruses in children admitted to an infant/toddler unit improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs what is the role of respiratory viruses in community acquired pneumonia; what is the best therapy for influenza and other viral causes of cap? detection of human bocavirus and human metapneumovirus by real-time pcr from patients with respiratory symptoms in southern brazil influenza burden in febrile infants and young children in a pediatric emergency department laboratory diagnosis, epidemiology, and clinical outcomes of pandemic influenza a and community respiratory viral infections in southern brazil respiratory viral infections in infants: causes, clinical, symptoms, virology, and immunology viral respiratory infection in curitiba, southern brazil the first wheezing episode: respiratory virus etiology, atopic characteristics, and illness severity who. cdc protocol of real time rtpcr for influenza a (h n ) viral infections in outpatients with medically attended acute respiratory illness during the - influenza season key: cord- -m uha qn authors: daleno, cristina; piralla, antonio; scala, alessia; senatore, laura; principi, nicola; esposito, susanna title: phylogenetic analysis of human rhinovirus isolates collected from otherwise healthy children with community-acquired pneumonia during five successive years date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: m uha qn in order to evaluate the circulation of the different human rhinovirus (hrv) species and genotypes in italian children with radiographically confirmed community-acquired pneumonia (cap), a nasopharyngeal swab was obtained from children admitted to hospital because of cap during five consecutive winter and early spring seasons ( - ). real-time reverse transcriptase polymerase chain reaction (rt-pcr) was used to identify hrv, and the hrv-positive samples were used for sequencing analysis and to reconstruct the phylogenetic tree. hrv was identified in samples ( . %), and the vp /vp region was successfully amplified in ( . %). hrv-a was identified in samples ( . %), hrv-b in ( . %) and hrv-c in ( . %). forty-seven ( . %) of the children with hrv infection were aged < year, ( . %) were aged - years, and ( . %) were aged ≥ years. blast and phylogenetic analyses showed that the hrv strains were closely related to a total of reference genotypes, corresponding to hrv-a, hrv-b and hrv-c strains. nucleotide variability was % between hrv-a and hrv-b, . % between hrv-a and hrv-c, and . % between hrv-b and hrv-c. a number of sequences clustered with known serotypes and, within these clusters, there were strains circulating during several seasons. the most frequently detected genotypes were hrv-a (n= ), hrv-a (n= ) and hrv-c (n= ). this study shows that, although it is mainly associated with hrv-a, pediatric cap can also be diagnosed in subjects infected by hrv-c and, more rarely, by hrv-b. moreover, a large number of genotypes may be involved in causing pediatric cap and can be different from year to year. although the prolonged circulation of the same genotypes can sometimes be associated with a number of cap episodes in different years. the use of molecular methods of respiratory viral screening has recently made it possible to establish that human rhinoviruses (hrvs) are not only the main cause of the common cold (as thought since they were first identified several decades ago), but also common etiologic agents of lower respiratory tract infections (lrtis) such as bronchiolitis and community-acquired pneumonia (cap) [ ] [ ] [ ] [ ] [ ] [ ] . it has also been reported that they are the pathogens most frequently associated with asthma exacerbations [ , ] . these findings have significantly increased interest in better defining hrv epidemiology mainly in order to clarify the possible association between specific strains and the development of more severe respiratory problems. a number of studies have reported epidemiological data showing hrv infection rates in children and adults [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it has been generally found that various hrv genotypes simultaneously circulated in a given period of time in a specific geographic area, most of which belonged to the a and c species; furthermore, the genotypes belonging to species a were mainly associated with cap and those belonging to species c were mainly associated with wheezing. however, most of these studies were carried out in a single year and involved a relatively small number of patients, and only a few analysed specific lrtis. consequently, there are few data concerning the circulation of hrvs over a long period of time or the real role of the different species and genotypes in causing lrtis. the aim of this study was to evaluate the circulation of the different hrv species and genotypes in italian children with radiographically confirmed cap during the winter and early spring of five consecutive years as this information could help to develop tailored strategies for the prevention and treatment of pediatric hrv infections. the study was carried out in pediatric clinic of the department of pathophysiology and transplantation of the university of milan during five consecutive years. the enrolment occurred between november and april in the years - , - , - and - and between november and june in - . it was approved by the institutional review board of the fondazione irccs ca' granda, ospedale maggiore policlinico, milan, italy. the written informed consent of a parent or legal guardian was required, and the older children were asked to give their assent. all of the children aged between one month and years seen in the emergency room (er) of the department of pathophysiology and transplantation who had fever (defined as an axillary temperature of > °c), signs and symptoms of lower respiratory tract infection (i.e. cough, tachypnea, dyspnea or respiratory distress, and breathing with grunting or wheezing sounds with rales) and a chest radiograph consistent with cap (in accordance with the world health organization criteria for the standardized interpretation of pediatric chest radiographs for a diagnosis of pneumonia [ ] ) were considered eligible for the study. the exclusion criteria were chronic diseases increasing the risk of respiratory infections, including premature birth; chronic disorders of the pulmonary or cardiovascular systems, including asthma; chronic metabolic diseases, including diabetes mellitus; neoplasia; kidney or liver dysfunction; hemoglobinopathies; immunosuppression; diseases requiring long-term aspirin therapy; and genetic or neurological disorders. the children with presumed nosocomial cap (i.e. appearing more than hours after admission or within two weeks of hospital discharge) were also excluded. a sample of nasopharyngeal secretions was collected from all of the children with radiographically confirmed cap using a flexible pernasal flocked swab that was immediately placed in a mini-tube containing ml of universal trasport medium (utm-rt kit cat. no. c, copan italia, brescia, italy). viral nucleic acids were extracted from the nasopharyngeal swabs using a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extracts were tested for respiratory viruses using the rvp fast assay (luminex molecular diagnostics inc., toronto, canada) in accordance with the manufacturer's instructions. the rvp fast assay consists of a single multiplex polymerase chain reaction (pcr) with labelled primers, followed by the single-step hybridization of the pcr products with the fluorescent bead array and incubation with reporter reagents. the plate was analysed using a bio-plex system (bio-rad laboratories, milan, italy) and its associated software luminex x ponent version . (luminex molecular diagnostics inc., provided by abbott), and the median fluorescent intensity (mfi) was determined. an mfi above the threshold level determined by the manufacturer for a particular target indicated a positive result for that target. the mean positive fluorescence intensities were established using tag-it data analysis software (tdas, luminex). the rvp fast assay simultaneously detects influenza a virus (subtyped h or h ), influenza b virus, respiratory syncytial virus (rsv)-a and -b, parainflunzavirus- , - , - and − , adenovirus, human metapneumovirus (hmpv), coronaviruses e, nl , oc and hku , enterovirus/ rhinovirus, and human bocavirus. the samples that were positive for enterovirus/rhinovirus were retested by means of one-step real-time rt-pcr in order to identify the rhinovirus [ ] . the hypervariable part of the ' ncr (non-coding region), the entire vp gene and the ' terminus of the vp gene in the hrv-positive samples were amplified by means of a rt-pcr as previously described [ , ] . the pcr products were purified using the wizard sv gel and pcr clean-up system (promega, milan, italy), and then sequenced in both directions using the same forward and reverse primers as those used in the pcr. the nucleotide sequences were obtained by means of automated dna sequencing using an abi prism genetic analyser (applied biosystems, foster city, ca). the newly determined sequences were edited using sequencher software, and aligned using the clustalw program integrated in the mega version . package [ ] . the analysed fragment was nt in the vp /vp region. the hrv in each sample was determined on the basis of the phylogenetic tree and by comparing it with all of the available rhinovirus reference prototypes encoding vp /vp protein retrieved from genbank (http://www.ncbi.nlm.nih.gov). the phylogenetic tree was reconstructed using the neighbour-joining method and parameters selected by the mega model test program. branch support was assessed by means of bootstrap analysis with replicates. genotypes were assigned on the basis of their clustering with known prototype reference strains present in genbank. the genetic distances between and within hrv-a, hrv-b, hrv-c were tested using the mega program and compared by means of student's t-test. the graphs were made using graphpad prism version . for windows (graphpad software, san diego, ca). the vp /vp sequences described in this study have been deposited in genbank under accession numbers kf -kf . during the five years of the study, children with radiographically confirmed cap were enrolled. of these, ( . %) were positive for at least one virus, and ( . %) were negative. hrv was identified in samples ( . %), in % of the cases as single infectious agent. the vp /vp region was successfully amplified in ( . %); the remaining samples ( . %) were excluded from the phylogenetic analysis because of poor sequence quality. all three hrv types were circulating in the study population: type hrv-a was identified in samples ( . %), hrv-b in ( . %), and hrv-c in ( . %). as shown in table , ( . %) of the children with hrv infection were aged < year, ( . %) were aged - years, and ( . %) were aged ≥ years. hrv-a and hrv-c were equally frequent in the patients aged < year ( . % vs . %), whereas their incidence was different in the children aged ≥ years ( . % vs . %). hrv-b was the less frequently species in all three age categories. figure shows the monthly distribution of the hrv species over the -year study period. hrv-a and hrv-c was circulating in almost all of the months of surveillance without any clear seasonal pattern: hrv-a cases were frequently observed in february ( / ; . %) and march ( / ; . %), whereas hrv-c cases were frequently observed in december ( / ; . %). hrv-b occurred only sporadically throughout the study period. on the basis of blast and phylogenetic analyses, the italian hrv strains were closely related to a total of reference genotypes corresponding to hrv-a, hrv-b and hrv-c strains. table shows the frequencies of the hrv genotypes detected in each season during the study period. the hrv sequences showed marked genetic diversity. the hrv-c sequences were the most heterogenous, with an intraspecies nucleotide p-distance of . , which was greater than the p-distance within hrv-a ( . ) or hrv-b ( . ). nucleotide variability was % between hrv-a and hrv-b, . % between hrv-a and hrv-c, and . % between hrv-b and hrv-c. figure shows the phylogenetic tree constructed on the basis of the vp /vp region of the sequences from this study and the references sequences. a number of sequences clustered with known genotypes and, within these clusters, there were strains circulating during hrv-a -like strains were observed during three alternate seasons ( - , - , - ) . the italian hrv-a sequences seem to cluster into two different groups (a and b, figure a ): in comparison with the reference strain, mean nucleotide divergence in cluster b was significantly greater than in cluster a ( . % vs . %; p< . ). the hrv-a strain circulated in four consecutive seasons ( - ). six of the nine hrv-a -like sequences ( . %) showed high degree of identity (mean %) in comparison with that between them and the reference strain (mean %); the remaining three strains were dispersed with hrv-a -like strains circulated worldwide (fig. b) . it is worth noting that two identical hrv-a strains were observed in two different seasons (mi -jan and mi -dec ), and the same strains were also found in the usa in , and italy and switzerland in . hrv-a -like strains clustered in two different groups with respectively . % and . % nucleotide divergence from the reference strain. these sequences circulated in two nonconsecutive seasons ( - and - ); however, one strain found in - (mi ) showed a greater degree of nucleotide similarity with two hrv-a -like sequences circulating in - than with another strain circulating in the same year. this could mean that the hrv-a -like strains remained in the studied population for a long time, probably more than two years ( figure c ). an identical hrv-a -like strain was identified in samples taken during two non-consecutive seasons (mi -nov and mi -feb ). in addition, a series of hrv-a-like sequences showed a nucleotide divergence of > . % from their reference strains: mi and mi vs hrv-a , and mi and mi vs hrva- (figure a ). all of the sequences defined as being hrv-b -like and hrv-b -like strains showed a nucleotide divergence of > . % from the reference strains (mean . % and . %) ( figures d and e ). hrv-b -like strains were the most frequent b genotype, and circulated in three different seasons. hrv-b like strains were found in two different seasons with the presence of the same strain in both years (mi -dec and mi -apr ), and a nucleotide divergence of . % from the reference strain. two strains (mi -mar and mi -dec ) clustered alone without any reference strains ( figure b ). the nucleotide identity of the two sequences with hrvc pat (eu , which is not currently assigned to genotypes) was > % (figure f) . the four hrvc- -like strains were all observed in december of three different seasons. two of them (mibr -dec and mibr -dec ) showed significantly greater nucleotide divergence from the reference strain than the other table . hrv genotypes within each species detected among study subjects. species and genotypetotal - - - - - two strains (mi -dec and mi -dec ) (mean . % and . %; p< . ), which suggests that they may belong to a new hrv-c genotype ( figure g ). however, the sequencing of vp for these two strains is required for type assignment. the mi -dec and mi -dec sequences showed more than . % nucleotide divergence ( . % and . %, respectively) from their most similar reference strains (hrv-c and hrv-c ). these two sequences respectively grouped with the unassigned serotype sequences hrvc pat (eu ) and hrvc pat (fj ) ( figure b ). this study is based on five years' surveillance of hrv infection in italian children with radiographically confirmed cap and confirms the high frequency of the association between this virus and pediatric cap. the presence of a virus in the nasopharynx of a child with cap does not necessarily mean that it is the etiological agent of the disease because it may only indicate a coincidental upper airways infection, or be due to a carrier state or the prolonged shedding of a pathogen that caused a previous infection. this may be particularly important in the case of rvs because a number of epidemiological studies have shown that they can be found in the respiratory secretions of - % of asymptomatic subjects [ , ] . however, in our study population, we found hrv as a single agent in about % of the children in which this pathogen was identified. this strongly suggests that it was the cause of the cap diagnosed in many of our study patients, particularly in those in whom very low values of wbc counts and crp were found. the most frequently detected hrv species was hrv-a ( . %), followed by hrv-c ( . %) and hrv-b ( . %). species and genotypetotal - - - - - these detection rates are similar to those recently reported in various countries [ ] [ ] [ ] , but different from those of calvo et al. [ ] and linsuwanon et al. [ ] , who studied patients with lrtis and found that hrv-b was predominant when cap was diagnosed. however, the diagnosis of cap in the last two studies was not always radiographically confirmed and this could have affected the results. we detected hrvs more frequently in children aged < year ( . %) and in those aged - years ( %), than in those aged ≥ years ( . %), as has been reported in other studies [ , ] . in terms of species, hrv-a was more frequently detected than hrv-c in children aged - and ≥ years, although a recent study in thailand found that hrv-c was the most frequent species in children with lrtis in the same age groups [ ] . in our study the circulation of hrv was evaluated only during the winter and spring months. this means that data do not indicate the real circulation of hrv species during the whole year. however, ours is the first study defining the circulation of hrv species and genotypes in children with cap that is based on such a large number of samples collected over a -year observation period, which is significantly longer than in other studies [ , [ ] [ ] [ ] [ ] and can give a precise information on the circulation in time of the different hrv species. . the hrv-positive samples contained a total of genotypes ( hrv-a, nine hrv-b and hrv-c), which is quite similar to the number reported in a recent -year study of children and adults with lrtis in sweden [ ] , but higher than the genotypes found in a study carried out in central italy [ ] . however, the observation period of this study was limited to one year and only a small number of cap patients were enrolled. we classified the hrv genotypes using vp /vp -based phylogenetics, and distinguished the inter-and intra-serotypes using the nucleotide divergence threshold very recently proposed by mcintyre et al.: . % for hrv-a, . % for hrv-b, and . % for hrv-c [ ] . however, in the absence of a vp sequence, the putative new types are considered provisionally assigned. our data show that a large number of genotypes can circulate during each observation period, and that some can circulate in more than one year and simultaneously in different geographic areas. this is clearly shown by the similarity between the italian hrv-a strains identified in the present study and those found in spain [ ] and jordan [ ] during , and the fact that we also found the genotypes most frequently detected in sweden by sansone et al.: a , a , a , a , c and c [ ] . considering only the - season, genotypes a , a , a and c found in this study were also found in a study carried out in france and involving a population similar to ours [ ] . furthermore, hrv-a was the most common genotype found in children with a primary diagnosis of viral cap in beijing, china, during the - season [ ] . there was a wide range of genotypes with different degrees of nucleotide variations from their reference strain. increased nucleotide variability with respect to prototype strains has been observed in studies carried out all over the world [ , , , [ ] [ ] [ ] . the considerable attention given to sequencing over the last few years and the development of efficient molecular methods capable of characterising hrv species and genotypes will allow the discovery of new variants in the near future. our findings show that, although cap is mainly associated with hrv-a, it can also be diagnosed in subjects infected by hrv-c and, more rarely, hrv-b. our analysis of the genotypes associated with pediatric cap suggests that a large number of genotypes may be involved in causing the disease. these may vary from year to year although the prolonged circulation of the same genotypes can sometimes be associated with a number of cap episodes in different years. this could be the case of hrv-a -like and hrv-a -like strains, which were the most frequently found in this study. however, the importance of these types and the need to develop preventive measures against them should be confirmed in further studies of larger populations from different parts of the world. further studies are also needed to evaluate whether any of the different types of hrv is more frequently associated with bacterial superinfection. finally, it could be useful to analyse their ability to cause viremia, which has been recently associated with more severe hrv disease [ ] . conceived and designed the experiments: se np. performed the experiments: cd as. analyzed the data: ap. contributed reagents/materials/analysis tools: cd as ls np se. wrote the manuscript: cd ap np se. pathogenesis of rhinovirus infection molecular diagnosis of respiratory virus infections pneumonia in pediatric outpatients: cause and clinical manifestations rhinovirus infection in hospitalized children in hong kong: a prospective study impact of rhinoviruses on pediatric community-acquired pneumonia impact of viral infections in children with community-acquired pneumonia: results of a study of respiratory viruses rhinovirus and the lower respiratory tract new developments in the epidemiology and clinical spectrum of rhinovirus infections lower respiratory infections among hospitalized children in new caledonia: a pilot study for the pneumonia etiology research for child health project role of emerging respiratory viruses in children with severe acute wheezing detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study is respiratory viral infection really an important trigger of asthma exacerbations in children? standardization of interpretation of chest radiographs for the diagnosis of pneumonia in children genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods predominance of rhinovirus in the nose of symptomatic and asymptomatic infants comparison of results of detection of rhinovirus by pcr and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness human rhinovirus causes severe infection in preterm infants molecular epidemiological study of human rhinovirus species a, b and c from patients with acute respiratory illnesses in japan prospective genotyping of human rhinoviruses in children and adults during the winter of - role of rhinovirus c respiratory infections in sick and healthy children in spain high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease rhinovirus-associated hospitalizations in young children human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander clinical severity and molecular typing of human rhinovirus c strains during a fall outbreak affecting hospitalized patients phylogenetic patterns of human respiratory picornavirus species, including the newly identified group c rhinoviruses, during a -year surveillance of a hospitalized patient population in italy genetics, recombination and clinical features of human rhinovirus species c (hrv-c) infections; interactions of hrv-c with other respiratory viruses rhinovirus infections in western sweden: a four-year molecular epidemiology study comparing local and globally appearing types proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types human rhinovirus a isolate so polyprotein gene genbank fj . human rhinovirus sp. isolate polyprotein gene human rhinovirus c infections mirror those of human rhinovirus a in children with community acquired pneumonia evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection screening respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp -vp typing reveals high incidence and genetic diversity of hrv species c phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children key: cord- -ddpud b authors: waghmare, alpana; xie, hu; kuypers, jane; sorror, mohamed l.; jerome, keith r.; englund, janet a.; boeckh, michael; leisenring, wendy m. title: human rhinovirus infections in hematopoietic cell transplant recipients: risk score for progression to lower respiratory tract infection date: - - journal: biol blood marrow transplant doi: . /j.bbmt. . . sha: doc_id: cord_uid: ddpud b human rhinovirus lower respiratory tract infection (lrti) is associated with mortality after hematopoietic cell transplantation (hct); however, risk factors for lrti are not well characterized. we sought to develop a risk score for progression to lrti from upper respiratory tract infection (urti) in hct recipients. risk factors for lrti within days were analyzed using cox regression among hct recipients with rhinovirus urti between january and march . the final multivariable model included factors with a meaningful effect on the bootstrapped optimism corrected concordance statistic. weighted score contributions based on hazard ratios were determined. cumulative incidence curves estimated the probability of lrti at various score cut-offs. of rhinovirus urti events, ( %) progressed to lrti. in a final multivariable model allogeneic grafts, prior rhinovirus urti, low lymphocyte count, low albumin, positive cytomegalovirus serostatus, recipient statin use, and steroid use ≥ mg/kg/day were associated with progression to lrti. a weighted risk score cut-off with the highest sensitivity and specificity was determined. risk scores above this cut-off were associated with progression to lrti (cumulative incidence % versus % below cut-off; p < . ). the weighted risk score for progression to rhinovirus lrti can help identify and stratify patients for clinical management and for future clinical trials of therapeutics in hct recipients. human rhinovirus (hrv) is the most commonly detected respiratory virus after hematopoietic cell transplantation (hct) in both the upper and lower respiratory tract [ , ] . although rare, once virologically confirmed lower respiratory tract infection (lrti) has developed, mortality rates due to hrv appear to be high ( % to %) and even similar to those seen with other respiratory viruses including influenza, respiratory syncytial virus (rsv), and parainfluenza viruses (pivs) [ ] [ ] [ ] . risk factors associated with lrti in this population have been defined for rsv, pivs, influenza, and human metapneumovirus (hmpv) [ ] [ ] [ ] [ ] [ ] , which include lymphopenia, use of highÀdose steroids, and conditioning regimen; this has not been done systematically in hct recipients presenting with hrv upper respiratory tract infection (urti). in a study of hrv infection that included both hct recipients and patients with hematologic malignancies, factors more likely to be present in patients with lrti included inpatient status, lymphopenia, and hypoalbuminemia [ ] . of note, not all cases of lrti were virologically confirmed in this study, and the analysis was not conducted in a timeÀdependent manner. furthermore, several key factors including viral load were not evaluated in this study. in the present study we aimed to identify specific risk factors for progression to lrti in a cohort of patients presenting with urti. we then developed a risk score for disease progression to help clinicians risk stratify patients who may benefit from more intensive monitoring and follow-up. ultimately, identification of highÀrisk patients will be important for clinical trial design that will require defining an enriched cohort at high risk for lrti that may benefit from potential interventions. research center. subjects were eligible for inclusion in the study if positive for hrv from the upper respiratory tract post-transplant. patients with hrv infection before transplant were excluded. clinical data were collected from databases and supplemental review of the medical record. this study was approved by the institutional review board at the fred hutchinson cancer research center. subjects signed informed consent permitting the use of data for research. respiratory tract samples collected as part of routine clinical care from adult and pediatric patients with respiratory symptoms were tested for respiratory viruses by real-time reverse-transcriptase pcr (rt-qpcr) assays, including rsv, hmpv, influenza viruses a and b, pivs to , adenovirus, human coronaviruses, hrv, and human bocavirus. rt-qpcr with this multiplex panel was performed on all respiratory samples during the time period of this study. the rt-qpcr cycle threshold (ct) was used as a proxy for viral load, with lower ct indicating a higher viral load. ct values were analyzed in quartiles as well as above and below the median. urti was defined as a positive hrv pcr result from an upper tract sample (nasopharyngeal swab or wash) only from a symptomatic patient, without any radiographic abnormalities. lrti was defined as a positive lower tract sample (bronchoalveolar lavage [bal], lung biopsy, or autopsy specimen) with or without radiographic abnormality (proven or probable lrti, respectively) or a positive upper tract sample with radiographic abnormality (possible lrti). both chest x-rays and chest computed tomography results were included if completed within weeks of last positive hrv date. lower tract sample results were included up to months from urti date. the day of lrti diagnosis was defined as the date when hrv was detected in the lower tract specimen (in proven/probable lrti) or the date the radiographic abnormality was detected (in possible lrti). patients who met criteria for lrti within days of urti were considered to have lrti at presentation and were not included in the progression analyses. an hrv illness event was considered to be a new event if weeks elapsed between positive samples or if there were negative hrv samples between hrv positive samples. the probability of progression from upper to lower hrv infection was estimated using cumulative incidence, treating death as a competing risk event. factors associated with progression to lrti were evaluated using cox regression models. the following variables were considered potential predictors of progression: patient age; sex; donor type; cell source; year transplant performed; conditioning regimen; most recent wbc count; neutrophil, lymphocyte, monocyte, and platelet counts before hrv infection (within weeks); lowest albumin level in the weeks before hrv infection; highest daily steroid dose in the weeks before hrv infection; pretransplant lung function; hct-specific comorbidity index (hct-ci) [ ] ; time from transplant to hrv infection; hrv ct values, acute and chronic graft-versus-host-disease; and recipient and donor cytomegalovirus (cmv) serostatus. recipient and donor statin use was defined as previously reported [ ] . all covariates with p . in the univariable analyses were candidates for inclusion in the multivariable cox regression models. initially, any variable was retained in the multivariable model if its p . in the full model. the multivariable model was further reduced for constructing the risk score by evaluating the change in the bootstrapped optimism corrected concordance statistic (c-statistic) when each factor was removed from the full model [ ] . the bootstrapped c-statistic was based on replications. those factors having little impact on the c-statistic were removed. score point contributions were developed using the hazard ratios from the final multivariable model. hazard ratios were rounded to the nearest integer and scaled such that the maximum achievable score was . the risk score for each subject was calculated based on their covariate values and the associated points given. curves were constructed to examine impact of shifting cut points of the risk score on sensitivity and specificity rates. cumulative incidence curves for progression to lrti for score values above (positive predictive value) and below (negative predictive value) a series of binary cut-off values as well as within categorized ranges of scores were evaluated and compared using log-rank tests and gray's test. subjects with missing values for albumin, cmv serostatus, and recipient statin status were excluded from univariable and multivariable analyses. statistical significance was defined as -sided p < . . sas version . ts m (sas institute, inc., cary, nc) and r version . . [ ] were used for statistical analyses. among patients undergoing hct during the study time period, ( %) were positive for hrv from at least respiratory site (figure ), resulting in hrv illness events. of these, events ( %) represented urti at presentation, whereas % of events involved presentation with lrti. of urti events, events had complete data for all covariates and were included in the progression analyses. among subjects with multiple hrv urti events, the median time between events was days (interquartile range [iqr], to ). in subjects with urti at presentation, median age was . years (iqr, . to . ), and there was a slight male predominance ( %) ( table ). seventeen percent of urti events ( / ) progressed to lrti with ( %) proven lrti events and ( %) possible lrti events. no probable lrti events occurred. most hrv urti events occurred in recipients of allogeneic hct ( / ; %); % ( / ) of events progressed to lrti in this group with ( %) proven lrti events and ( %) possible lrti events. median time to progression from urti to lrti was days for all events (iqr, to ) and days for allogeneic subjects only (iqr, to ) (figure a ,b). of subjects with possible lrti events, none had a bal performed. in univariable analysis of all events, donor type, conditioning regimen, prior hrv events, steroid use, albumin level, recipient statin use, recipient cmv serostatus, and most recent wbc count, and neutrophil, lymphocyte, and monocyte counts were sufficiently associated with progression to lrti to be considered for inclusion in a multivariable model (table ) . notably, the presence of viral co-pathogens, pulmonary function, or intravenous immunoglobulin, and hrv ct values in the upper tract sample were not associated with progression to lrti. univariate and multiple multivariate models for risk factors for progression to proven lrti only are shown in supplementary tables and . candidate variables from univariable analysis were evaluated in a multivariate model. covariates were added sequentially and c-statistic calculated for each model until the optimal model with the highest c-statistic was achieved (table ). in the final model the c-statistic was . , whereas the optimism corrected c-statistic was . . a risk score was developed as described and weighted contributions of each factor are shown in table . specificity and sensitivity of score cutÀoffs for development of lrti in the entire cohort and the allogeneic cohort are shown in figure a and b, respectively. among all subjects the score cut-off with the highest sensitivity ( %) and specificity ( %) was , corresponding to a positive predictive value of % and a negative predictive value of % in this population. cumulative incidence curves for progression to lrti in all subjects, stratified by above and below a range of binary score cutÀoffs are shown in figure a and b, representing positive and negative predictive values for a given cutÀoff, respectively. cumulative incidence of progression to lrti for the group was % versus % in the < group (log-rank and gray's test both p < . ). figure c illustrates the ability of the score to stratify the population into mutually exclusive levels of risk. cumulative incidence of progression to lrti was as follows: scores < ( %), to < ( %), to < ( %), and ( %) (log-rank test and gray's test both p < . ). the weighted risk score was applied to the allogeneic subgroup only and cumulative incidence curves above and below the cutÀoff are shown figure a . for scores the cumulative incidence was % versus % for scores < (logrank test and gray's test p < . ). the score was also applied to the whole cohort and evaluated with proven lrti only as the outcome. figure b shows the cumulative incidence curves above and below for progression to proven lrti only. for scores the cumulative incidence was % versus % for scores < (log-rank test and gray's test p = . ). in this large retrospective study of hct recipients infected with hrv in the upper respiratory tract, we established risk factors and developed a risk score for progression to lrti. to our knowledge this is the largest and statistically most rigorous study to systematically examine patients specifically presenting with urti to determine the risk of subsequent lrti. in multivariable models for any lrti event in all subjects, significant risk factors identified include allogeneic transplant, prior hrv urti events, lymphocyte count < £ cells/l, recipient statin use, recipient cmv serostatus, and steroid use mg/kg/day. we further characterized risk factors for progression by calculating the change in the bootstrapped optimism corrected c-statistic for each risk factor and developed an optimized model for risk prediction. using this model we developed a weighted score and describe the performance characteristics of the score at various cutÀoffs. the risk score presented in this article is a novel tool with potential to help risk stratify patients for clinical care and future clinical trials. hrvs have recently been recognized as significant pathogens that can cause severe disease and poor outcomes in certain high risk groups, including infants, pregnant women, asthmatics, hospitalized adults, and immunocompromised children and adults [ , , , , , ] . once hrv lrti has developed in hct recipients, mortality rates are comparable with those associated with rsv, influenza, and piv [ ] . in a study of hrv infection that included both hct recipients and patients with hematologic malignancies, factors more likely to be present in patients with lrti included inpatient status, lymphopenia, and hypoalbuminemia [ ] . in hct recipients alone from the same center, hypoalbuminemia, and isolation of respiratory co-pathogen were associated with lrti in multivariable models [ ] . these analyses, although not done in a time-dependent manner, are similar to findings in the present study, in which lymphopenia and hypoalbuminemia were associated with progression to lrti. we evaluated copathogens in the upper respiratory tract as a risk factor and did not find an association with lrti. lymphopenia has been associated with progression to lrti for other respiratory viruses including pivs, rsv, hmpv, and influenza [ , , , , , ] , and was also more common in hrv lrti subjects in a prior study [ ] . monocytopenia has not been evaluated systematically in prior respiratory virus progression studies, although there was a trend toward significance for hmpv [ ] , and monocytopenia was associated with mortality in hct recipients with hrv and rsv lrti [ , ] . the relative role of monocytes over lymphocytes in progression of disease is not understood, and most studies have not evaluated both cell types concurrently. a potential mechanism of inflammatory and immune modulation via hrv induced monocytic cell directed cxl and ifn-g release is plausible [ ] . steroid use is a well described risk factor for progression to lrti for many respiratory viruses [ , , , ] , and higher doses of mg/kg/day were associated with hrv lrti in the present study. of note, highÀdose steroids are no longer frequently used for graftÀversusÀhost disease treatment at our institution, as is reflected in the low frequency of use ( %) in this cohort. however, even lower dose steroids ( to mg/kg/day) showed a trend toward significance in the multivariable models, and this was included in the weighted score. other centers may have higher rates of highÀdose steroid use and should still be able to apply the score to their patient population. prior post-transplant hrv urti infection was associated with increased risk of lrti, an interesting and somewhat unexpected finding suggesting an effect of the cumulative burden of hrv infections, perhaps by mediating inflammatory responses that increase the likelihood of lrti development. for parainfluenza viruses, urti without lrti is associated with late airflow decline, and a similar pathway may be at play for hrv infections [ ] . although it is possible we overestimated new events, our definition of > weeks between events was chosen given our recent surveillance data showing the median duration of hrv shedding in hct recipients is . days (range, to ) [ ] . given the long duration between events in the present study (median, days; iqr, to ), we do not believe we are capturing prolonged viral shedding but rather truly new viral events. recipient cmv serostatus was associated with increased risk of lrti, a novel finding for respiratory virus disease severity. cmv has a profound impact on the immune system in both immunocompetent and immunosuppressed individuals and known to have immunosuppressive effects [ ] [ ] [ ] . it is well known to affect nonrelapse mortality in hct patients [ ] . cmv has also been shown to increase the risk of other viruses after hct; however, the mechanisms are poorly understood. that cmv was associated with progression in this study is plausible but the results should be validated in a different cohort, and further studies are needed to elucidate the mechanism. recipient statin use as a risk factor for hrv lrti is also an intriguing finding and corroborates prior results from a separate cohort at our center that demonstrated a trend toward association between recipient statin use and all respiratory viral infections and lrtis [ ] . statins may act as immunosuppressants via direct inhibition of induction of mhc-ii expression by ifn-g and thus leading to reduced t cell activation [ ] and may also have an effect of type i ifn receptor signaling in monocytes in the setting of hrv challenge, leading to reduced inflammatory response [ ] . the role of statins on hrv progression should be validated in other cohorts and evaluated for other respiratory virus infections independently. we evaluated the impact of hrv viral load and found no association with progression to lrti. ct value as a surrogate for viral load has limitations given the diversity of hrv genotypes and the resulting differences in amplification efficiency using rt-qpcr assays with a consensus hrv primer and probe set. rt digital pcr has been recently shown to provide more precise quantification [ ] . however, even when evaluating the extreme highest viral load group (ct values in the lowest th percentile), no effect was seen. in lung transplant recipients, higher hrv viral load was associated with increased symptoms associated with lrti [ ] . in immunocompetent pediatric patients, viral load was associated with disease severity in older but not younger children [ ] and was also associated with higher rates of viremia and oxygen use [ ] . the variable role of viral load in studies to date suggest both limitations in capturing accurate viral loads from clinical samples such as was done in our study and that other host factors including immune responses may be an important player in progression to lrti and other clinical outcomes. additionally, hrv species were not evaluated in the present study, and data have suggested hrv-c may be associated with worse outcomes, especially in children [ , ] . the presence of viral coÀpathogens in the upper respiratory tract was not associated with progression to hrv lrti. to provide clinical applicability for our findings, we developed weighted risk scores for progression to lrti using methodology that allows for multiple sampling within the dataset to develop an optimized model [ ] . our final model showed an acceptable but not robust c-statistic of . . using this model we developed a weighted score and show specificity and sensitivity calculations for a range of score cutÀoffs, as well as cumulative incidence curves stratified by score cutÀoffs and within mutually exclusive score ranges. maximum sensitivity and specificity was demonstrated at a score cutÀoff of ; however, optimal score cutÀoff depends on the ultimate application of the score. for example, if the risk score is used for patient risk stratification for interventional clinical trials, a score cutÀoff with higher sensitivity and negative predictive value may be desirable. sample size limited our ability to extensively evaluate proven lrti, which for other respiratory viruses (rsv and piv) has been associated with worse clinical outcomes [ , ] . in several separate multivariate models, however, risk factors for progression to proven lrti were similar (low albumin, low monocyte count, and highÀdose steroids). we applied our risk score to the proven lrti outcome only, and subjects with a risk score higher than were associated with proven lrti. with a larger sample size a separate risk score for proven lrti could be developed, although it is likely that risk factors would remain similar. probable lrti could represent contamination . cumulative incidence curves for progression to lrti in all subjects, stratified by above (a) and below (b) a range of binary score cut-offs, representing positive and negative predictive values for a given cut-off, respectively. log-rank and gray's test comparing the versus < groups both p < . . cumulative incidence curves for progression to lrti in all subjects, stratified into mutually exclusive levels of risk (c; log-rank test and gray's test p < . ). from the upper respiratory tract during the bal procedure; however, there were no cases of probable lrti in our cohort. although our analyses used an accepted resampling methodology (bootstrapping), ultimately a multicenter study is needed for validation of the score, especially given differences in clinical practices including the use of highÀdose steroids. an immunodeficiency scoring index was developed for allogeneic hct recipients with rsv infection to predict lrti and rsv associated mortality [ ] ; the score was also applied to influenza [ ] . similar variables were evaluated in the present study and were not associated with the outcome with the exception of steroid use, suggesting rhinovirus progression may be determined by a unique set of risk factors compared with other respiratory viruses and a separate risk score is needed. this study has strengths and limitations. first, this is a retrospective study in which data collection, including viral load determination, was dependent on clinical databases. however, at our institution data from transplant recipients is prospectively recorded in a central database, and we also conducted detailed chart review on every patient to obtain additional clinical data. second, we captured both proven lrti (hrv detected in the lower respiratory tract with abnormal imaging) figure . cumulative incidence curves of progression to lrti in allogeneic recipients only (a) and of progression to proven lrti in all subjects (b), stratified above and below score of . log-rank test and gray's test p < . for both curves. and possible lrti (hrv detected in the upper respiratory tract with abnormal imaging but no lower tract sampling). at our institution the use of bal is based on standardized protocols outlining that bal should be performed in hct recipients with a respiratory virus detected in the upper respiratory tract and abnormal chest imaging. however, ultimately the need for bal is determined by the attending physician, and thus some proven or probable cases (hrv detected in the lower respiratory tract without abnormal imaging) may have been missed. by including both proven and possible lrti events (no probable events occurred), this effect is mitigated; however, proven lrti may be the more relevant outcome given the mortality rates seen with proven hrv lrti and the differences in outcomes observed in proven lrti for piv and rsv [ , , ] . regardless, many institutions do not routinely use bal for virologic confirmation, and thus our results for any lrti events can be broadly applicable. finally, our data include both allogeneic and autologous hct recipients, and there may be a bias toward increased testing in the allogeneic group and therefore more lrti events captured. however, the risk score performed reasonably well in the allogeneic subgroup. we were not able to evaluate risk factors for hrv lrti cases without any coÀpathogens in the lower respiratory tract separately. in a prior study the presence of coÀpathogens was not associated with mortality in hct recipients with proven hrv lrti, suggesting that hrv detection in the lower tract is pathogenic regardless of coÀpathogens and therefore making any proven hrv lrti an important endpoint [ ] . because bal was not done in possible lrti cases, it is plausible that other pathogens are contributing to the radiographic changes, and thus these are not true lrti events. however, as stated above, in clinical practice bal is often not attempted, and coÀpathogen data are not available, yet patients are considered to have lrti. thus, we included possible lrti as an endpoint. several epidemiologic studies and clinical trials of respiratory viruses in hct recipients have used definitions of lrti that include possible cases [ , , , [ ] [ ] [ ] [ ] [ ] . in summary, we describe the risk factors and development of a risk score for progression to lrti in a large cohort of hct recipients with hrv urti. the risk score proposed may have implications for risk stratifying patients for more intense clinical monitoring. additionally, risk stratification will help inform the design of future randomized clinical trials of novel therapeutics, including the growing field of virus specific t cells, which are currently being evaluated for other respiratory viruses including piv and hmpv [ , ] . human coronavirus (hcov) and rhinovirus (hrhv) infection among hematopoietic stem cell transplantation (hct) recipients progression, shedding patterns, and clinical disease associated with respiratory virus infections after allogeneic hematopoietic cell transplantation human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients respiratory viral infections after bone marrow/peripheral stem-cell transplantation: the christie hospital experience respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review human metapneumovirus infections following hematopoietic cell transplantation: factors associated with disease progression differences in clinical outcomes after influenza a/h n and seasonal influenza among hematopoietic cell transplant recipients immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy hematopoietic cell transplantation (hct)-specific comorbidity index: a new tool for risk assessment before allogeneic hct the association between donor and recipient statin use and infections after allogeneic hematopoietic cell transplantation multivariable prognostic models: issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors r: a language and environment for statistical computing. vienna: austria; r found stat comput nosocomial transmission and genetic diversity of rhinovirus in a neonatal intensive care unit prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical parainfluenza virus type ab in allogeneic hematopoietic cell transplant recipients: factors influencing post-transplant ab titers and associated outcomes outcomes of influenza infections in hematopoietic cell transplant recipients: application of an immunodeficiency scoring index respiratory syncytial virus lower respiratory disease in hematopoietic cell transplant recipients: viral rna detection in blood, antiviral treatment, and clinical outcomes human monocytic cells direct the robust release of cxcl by bronchial epithelial cells during rhinovirus infection airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses initial high viral load is associated with prolonged shedding of human rhinovirus in allogeneic hematopoietic cell transplant recipients immunosuppressive effects of beta-herpesviruses variation in the human immune system is largely driven by non-heritable influences origin and evolution of the t cell repertoire after posttransplantation cyclophosphamide early cytomegalovirus reactivation remains associated with increased transplant-related mortality in the current era: a cibmtr analysis statins as immunomodulators simvastatin attenuates rhinovirusinduced interferon and cxcl secretion from monocytic cells in vitro superiority of digital reverse transcription-pcr (rt-pcr) over real-time rt-pcr for quantitation of highly divergent human rhinoviruses role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients rhinovirus load and disease severity in children with lower respiratory tract infections impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children clinical severity and molecular typing of human rhinovirus c strains during a fall outbreak affecting hospitalized patients human rhinovirus c in adult haematopoietic stem cell transplant recipients with respiratory illness supplemental oxygen-free days in hematopoietic cell transplant recipients with respiratory syncytial virus parainfluenza virus lower respiratory tract disease after hematopoietic cell transplant: viral detection in the lung predicts outcome impact of aerosolized ribavirin on mortality in allogeneic haematopoietic stem cell transplant recipients with respiratory syncytial virus infections an adaptive randomized trial of an intermittent dosing schedule of aerosolized ribavirin in patients with cancer and respiratory syncytial virus infection viral respiratory tract infections in allogeneic hematopoietic stem cell transplantation recipients in the era of molecular testing a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients a phase ii, randomized, double-blind, placebocontrolled study to examine the effects of das in immunocompromised subjects with lower respiratory tract parainfluenza infection on supplemental oxygen (das - - ) characterizing the cellular immune response to parainfluenza virus immunologic profiling of human metapneumovirus for the development of targeted immunotherapy presented in part at the bmt tandem supplementary data related to this article can be found online version at doi: . /j.bbmt. . . . key: cord- - gi wb authors: gambarino, stefano; costa, cristina; elia, mariateresa; sidoti, francesca; mantovani, samantha; gruosso, valentina; bergallo, massimiliano; cavallo, rossana title: development of a rt real-time pcr for the detection and quantification of human rhinoviruses date: - - journal: mol biotechnol doi: . /s - - -x sha: doc_id: cord_uid: gi wb human rhinoviruses (hrv) are the most common viral agents, being responsible for upper as well as lower respiratory tract infections. evidence demonstrating that hrv disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of hrv infections and the increasing number of immunocompromised patients underline the need for including hrv in virological diagnostics of acute lower respiratory tract illness. this article describes the development and optimization of a reverse transcription (rt) real-time pcr assay for quantification of hrv rna in clinical samples. efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. subsequently, the assay has been validated on bronchoalveolar lavage (bal) specimens obtained from immunocompetent and immunocompromised patients. pcr assays have markedly improved the diagnosis of human rhinoviruses (hrv) infections by increasing sensitivity in comparison to traditional cell culture [ ] ; however, many rt-pcr protocols still are time-consuming. real-time pcr assays have resulted in improving virus detection and quantification [ ] . rapidity and quantitative results represent important advantages in the diagnostic workup of hrv infection and could be of relevance considering the future potential availability of antiviral agents. human rhinoviruses are the most common viral agents in humans, being the over serologic types responsible for about - % of all the cases of common colds and associated upper respiratory tract complications in both adults and children. although hrv are generally temperature restricted in replication with optimal growth at - °c, the temperatures observed in the tracheobronchial tree are often lower than body core temperatures, also in relation to external temperature and frequency of ventilation, and so are permissive for hrv replication [ ] . moreover, it has been evidenced that many hrv serotypes can replicate efficiently at core body temperature with modest differences in terms of infectious titres (. - . log tcid ) [ ] . indeed, several studies have linked hrv infection to illnesses in the lower respiratory tract in immunocompetent patients, including exacerbations of preexisting airways disease in those with asthma, chronic obstructive pulmonary disease (copd) or cystic fibrosis, and pneumonia and bronchiolitis in children aged \ years hospitalized for hrv infection [ ] . however, a recent study on hospitalized adults with pulmonary disease found a prevalence of hrv in bronchoalveolar lavage (bal) specimens of . % without evidencing a significant difference of prevalence in relation to the presence of copd, pneumonia or acute respiratory illness [ ] . in contrast to immunocompetent subjects, hrv clearance in immunocompromised patients may be delayed with prolonged shedding leading to the onset of life-threatening conditions and chronic infections [ ] [ ] [ ] . evidence demonstrating that hrv disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of hrv infections and the increasing number of immunocompromised patients, underline the need for including hrv in virological diagnostics of acute lower respiratory tract illness. however, not many laboratories and institutions have adopted hrv diagnostics in the routine clinical use, and limited informations are available on hrv clinical impact in comparison with that of other respiratory viruses. the aim of this study was to develop and validate a rt real-time pcr assay for quantification of hrv rna in clinical samples. efficiency, sensitivity, specificity, interand intra-assay variability and dynamic range have been determined (fig. ). prototype rhinoviruses were obtained from the american type culture collection (atcc, manassas, va). clinical specimens originated from the virology unit of the fig. standard curve (from to copies/reaction) and dynamic range (from to copies/reaction) of the real-time rt-pcr developed in this study azienda ospedaliero-universitaria san giovanni battista, turin, and included bronchoalveolar lavages (bal) obtained from patients (m/f, / ; mean age, . years; range, . bronchoalveolar lavage procedure was performed as previously described [ ] for investigating the cause of unexplained fever, and/or respiratory symptoms, and/or new infiltrates on chest x-ray or for checking up a previous positivity or as routine follow-up in lung transplant recipients. before processing, specimens were thawed and liquefied with : n-acetylcisteine. clinical specimens were divided in two groups: (i) samples from automated extraction of rna was performed with the nuclisens easymag platform (biomeriéux, marcy l'etoile, france). rna was extracted from ll volume of clinical specimens, according to manufacturer's instructions, and eluted to a final volume of ll. the rt reaction was done in a separate tube by a random cdna syntesis from ll of extracted rna in two step: first a reaction mixture containing random primers ( ng/ ml)(invitrogen) and mm dntps (invitrogen, carlsbad, ca) was added and incubated at °c for min using the fast thermal cycler (applied biosystems, monza, italy); subsequently, a second reaction mixture containing . m dtt, buffer ( nm tris-hcl [ph . ], nm kcl), rnase out tm ( unit/ll)(invitrogen) and reverse transcriptase ( unit/ll)(superscript tm ii rt, invitrogen), giving a total volume ll, was used. after incubation for min at °c, min at °c and min at °c, the cdna mixture was used as target cdna. primers were designed within the highly conserved -utr region of hrv genome (table ) using the primer express . software (applied biosystems, cheshire, uk). briefly, ll of cdna was added to ll of pcr solution containing buffer , mm mgcl , unit recombinant subtherm dna polymerase (sic, rome, italy), lm of each dntp, lm of each sense and antisense primer. after an initial denaturation step of min at °c, the first-round pcr amplification was carried out under the following conditions: °c for s, °c for s, °c for s for cycles, then cycle at °c for min using the fast thermal cycler (applied biosystems). about ll of pcr product ( bp) was loaded onto a % (w/v) agarose gel containing . lg/ml ethidium bromide. the gel was visualized on a uv board. the pcr product was cloned using ptopo-ta cloning (invitrogen). recombinant plasmid was propagated in top high-efficiency chemically competent cells (invitrogen) using the heat shock procedure. after overnight culture, selected transformed clones were amplified by culture in liquid lb medium ( g bactotryptone, g yeast extract, mm nacl, g/l agar, ph . ) containing lg/ml of ampicillin. plasmid dna was extracted and purified using the wizard plus sv miniprep dna purification system (promega, madison, wi). the plasmid concentration was estimated on spectrophotometric reading at od . the dna copy number of the target plasmid was calculated from the molecular weight of the construct ( , , mw) and avogadro number ( . ). about ll of target plasmid contained . dna copy number. to determine the sensitivity of the pcr assays, stock preparations were diluted to - copies/ll by a series of -fold dilutions. the limit of detection is defined the lowest concentration of target quantified, whereas the efficiency of rt-pcr assay was also evaluated and is defined as (- /slope) [ ] . negative controls with rnase-free sterile water (sigma) were included in each batch of test samples for checking up carry-over contamination. negative controls: a number of precautions were undertaken to prevent the occurrence of false-positive results. each run included control reactions lacking template (notemplate controls) to test for the presence of contamination or the generation of nonspecific amplification products under the assay conditions used. positive controls: the presence of cellular gapdh transcript was analysed as internal control and marker of sensitivity of the assay; primer sequences for gapdh were designed using the software oligoperfect tm designer (invitrogen) within exon (forward primer) and exon (reverse primer) and are reported in table . the coefficient of variation for samples quantified in repeated measurements was calculated. descriptive statistics were expressed as absolute value, percentage and median as appropriate. the chi-squared test was applied to analyze results in clinical specimens. statistical analysis was performed using a commercially available software (med calc version . . . ). a p-value . was considered statistically significant. these parameters were assessed by repeated testing of serial logarithmic dilutions of the plasmid standard (covering a range of logs from to ). the number of genome copies in the plasmid preparation used for dilutions was determined by spectrophotometry at nm. after pcr amplification, the ct value (crossing point of the amplification curve with the pre-set threshold of fluorescence detection) of individual dilution steps was plotted against the initial virus copy number, leading to a typical standard curve. the amplification efficiency, defined by the standard curve slope, was generally between three and four. the consistency of replicates was measured by the correlation coefficient (r ), which indicates the linearity of the ct values plotted in the standard curves. the r index was higher than . . the actual sensitivity of assay was determined by the lowest standard dilution consistently detectable in replicate reactions at a frequency of % and it was found to be of copy/reaction and was linear (quantifiable) up to copies. a standard dilution of - copy/reaction was detected at a frequency of %, while that of - copy/reaction at a frequency of %. the limit of detection was - copy/reaction. the efficiency of the reaction is considered acceptable if it falls between the range of . and . with being ideal. the efficiency of our real-time rt-pcr assays was . . the reproducibility was expressed as the coefficient of variation (cv) in the log values of the concentration. furthermore, intra-and inter-assay variability was evaluated over different concentrations ranging from to plasmid copies within a single run (n = ) or different run experiments (n = ) by cv value of the ct and is reported in table . quantification of hrv was highly reproducible for as few as copies. the variability was studied by analysing samples tested in duplicate in five experiments. the results of the quantification revealed that the median ( th, th) cv was . % ( . - . %). the analytical specificity of the rt-pcr assay was determined by testing coxsackievirus types b (atcc vr- to examine the dynamic range of hrv rna quantification by rt-pcr, serial dilutions of the plasmid standard ranging from to copies/reaction were carried out. the rt-pcr assay was able to quantify hrv rna from to copies/reaction with a dynamic range of - copies/reaction (r = . ) obtained from linear regression analysis, without having to dilute high load samples. viral loads in clinical specimens were investigated in duplicate reactions with the appropriate external standard preparations. the efficiency of viral rna detection from clinical samples and the possible presence of polymerase inhibitors were monitored by using internal control, as described in the section 'materials and methods.' these controls permitted appropriate correction in the calculation of viral copy numbers. for the quantification of viral load, the slope (s) and the y-axis intercept (y) (the y-axis intercept is the point at which the standard curve intersects with the ordinate; it indicates the theoretical detection limit of the reaction by revealing the ct expected in the presence of a single target molecule in the sample) of the corresponding standard curve and the ct of the target virus amplification were used according to the following equation: po = inverse log(ct -y/s), where po is the number of virus copy equivalents in the pcr prior to amplification. results of hrv quantification are summarized in table . briefly, hrv was positive in / ( . %) bal specimens, obtained from as many patients ( / , . %): in particular / ( . %) samples from immunocompromised patients and / ( . %) samples from immunocompetent patients (p = n.s.). viral load was\ genome equivalents (geq)/ ml in the immunocompetent patient and ranged from \ to . geq/ml (median, ) in the immunocompromised patients. considering discharge diagnosis of pneumonia, respiratory insufficiency, exacerbations of preexisting bronchopneumopathies, other pneumopathies, there was no significant difference of prevalence between immunocompetent and immunocompromised patients. hrv was also detected in one sample obtained during table . this raticle describes the development of an in-house rt real-time pcr assay for quantification of hrv rna in clinical samples. real-time pcr assays are described as 'closed' systems, as no post-amplification manipulation of the amplicon is required. the advantages of these systems include a reduced turnaround time, minimisation of the potential for carry-over contamination and ability to closely scrutinise the assay performance, thus representing a suitable tool for routine diagnostics in virology. the use of an external standard curve for quantification relies upon titration of an identically amplified template, in a related sample matrix, within the same experimental run. however, this approach may suffer from uncontrolled and unmonitored inter-tube variations and thus such experiments should be described as semi-quantitative. despite this sub-optimal approach, fluorescence data are generally collected from pcr cycles that span the linear amplification portion of the reaction where the fluorescent signal and the accumulating dna are proportional. because the emissions from fluorescent chemistries are temperature dependent, data are generally acquired only once per cycle at the same temperature in order to monitor amplicon yield [ ] . then, the ct of the sample at a specific fluorescence value can be compared with similar data collected from a series of standards by the calculation of a standard curve. the determination of the ct depends upon the sensitivity and ability of the instrument to discriminate specific fluorescence from background noise, concentration and nature of the fluorescence-generating component and amount of template initially present. real-time pcr offers significant improvements to viral load quantification because of its wide dynamic range that can accommodate at least eight log copies of nucleic acid template [ ] [ ] [ ] . this is made possible because data are chosen from the linear phase of amplification where conditions are optimal, rather than the end-point where the final amount of amplicon could be affected by inhibitors, poorly optimised reaction conditions or saturation by inhibitory pcr by-products and double-stranded amplicon. the result of taking data from the end-point is that there may exist no relationship between the initial template and final amplicon concentrations. real-time pcr for the study of viral load presents a lower inter-and intra-assay variability in comparison to conventional pcr [ , ] and an equivalent or higher analytical sensitivity in comparison to traditional viral culture, or conventional single-round, and nested pcr [ ] . indeed, pcr methods have significantly improved the diagnosis of hrv infection. however, these reports could be an over-estimate due to the choice of smaller targets, which amplify more efficiently, or due to the use of different or improved primers for the real-time assays as the use of software to design optimised primers and oligoprobes is more common. when increased sensitivity and broad dynamic range are combined, it is possible to quantify template from samples containing a wide range of concentrations, as in clinical specimens. this avoids the need for dilution of the amplicon prior to conventional detection or repetition of the assay using a diluted sample because the first result falls outside the detection limits. the performance of the rt real-time pcr developed in this study was examined over different concentrations of hrv rna and it was found to be very sensitive with a minimum cut-off for detection of copy/reaction and was linear up to copies. the sensitivity, defined as the lowest concentration of hrv rna quantified at a frequency of %, was found to be copies/reaction. the assay reproducibility was high with a median inter-assay variability and intra-assay variability of . % (range . - . %) and . % (range . - . %), respectively, thus being superior to that obtained in other studies [ ] . the assay developed in this study was based on primers amplifying the -utr of the viral genome, that is highly conserved among entero-and rhinoviruses, and therefore, many pcr protocols amplify also enteroviruses. our assay resulted able to amplify hrv sequences, as confirmed by blast search, and should not amplify other viruses pathogenic to humans, in particular enteroviruses. other studies developed rt real-time pcr assays for detection of hrv [ , ] in respiratory specimens; however, our assay has been developed and optimized on the basis of the standard amplification profile for the applied biosystems real time pcr system, thus allowing the simultaneous processing of the same clinical specimens for different viruses potentially involved in respiratory tract infections. evaluation on clinical specimens confirmed the occurrence of hrv infection in the lower respiratory tract, as . % of bal specimens resulted positive. although prevalence of infection tended to be higher in specimens obtained from immunocompromised patients, the difference did not reach the statistical significance, as well as no significant association with a discharge diagnosis was found. regarding this, it has to be taken into account that all of our specimens, but one, presented a coinfection with at least another respiratory virus (including herpesviruses hsv, ebv, cmv, hhv- and - , human metapneumovirus, human coronavirus, human bocavirus, parainfluenza viruses, influenza viruses, adenoviruses, rsv)(data not shown). hrv shedding in the immunocompromised host could be prolonged in comparison to the immunocompetent one, thus leading to the onset of severe complications, as well as it has been suggested an association with acute rejection in lung transplant recipients [ ] . in our study, multiple sampling was available in particular for lung transplant recipients, however no repeated positivity was found, thus preventing the possibility to evaluate this issue and suggesting the need for further studies. similarly, no significant association was found with the occurrence of exacerbation of pre-existing bronchopneumopaties. as the association between acute exacerbations of asthma and chronic obstructive pulmonary disease and respiratory viral pathogens, particularly hrv, is well recognized [ ] [ ] [ ] , the lack of association found in our study could be due to the small number, thus preventing us to evaluate this issue. in conclusion, the rt real-time pcr assay developed in this study could represent a useful tool for diagnosing hrv infections, quantifying the viral load and could be applicable for routine diagnostic workup of upper as well as lower respiratory tract diseases. the availability of a quantitative approach for detection of hrv rna could represent a useful tool to investigate the epidemiological and clinical role on hrv in clinical contexts involving the immunocompromised hosts, in particular lung transplant recipients, or the association with exacerbations of preexisting bronchopneumopathies, in particular the relation between viral load and severity or duration of asthma exacerbations. molecular diagnosis of human rhinovirus infections: comparison with virus isolation real-time pcr for rapid diagnosis of enteroand rhinovirus infections using lightcycler thermal mapping of the airways in humans rhinoviruses replicate effectively at lower airway temperatures rhinovirus and the lower respiratory tract frequency of detection of respiratory viruses in the lower respiratory tract of hospitalized adults chronic rhinoviral infection in lung transplant recipient. american journal of respiratory and critical care medicine upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies what role for human rhinoviruses in the lower respiratory tract? new microbiologica quantitative detection of epstein-barr virus in bronchoalveolar lavage from transplant and non-transplant patients rapid cycle real-time pcr methods and application continuous fluorescence monitoring of rapid cycle dna amplification development and assessment of a novel real-time pcr assay for quantitation of hbv dna real-time reverse transcription-pcr for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children development of a sensitive real-time reverse transcriptase pcr assay with an internal control to detect and quantify chikungunya virus simultaneous detection of influenza viruses a and b using real-time pcr real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses amplicon sequencing and improved detection of human rhinovirus in respiratory samples role of viral infections in asthma and chronic obstructive pulmonary disease viral pathogens in acute exacerbations of chrnoic obstructive pulmonary disease virus infection, asthma, and chronic obstructive pulmonary disease acknowledgements we thank dr. paolo solidoro and dr. daniela libertucci, pneumology division of san giovanni battista hospital, turin, for their assistance in clinical sample analysis. key: cord- - w i t authors: de almeida, marina b.; zerbinati, rodrigo m.; tateno, adriana f.; oliveira, cristina m.; romão, renata m.; rodrigues, joaquim c.; pannuti, cláudio s.; da silva filho, luiz vicente f. title: rhinovirus c and respiratory exacerbations in children with cystic fibrosis date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: w i t to investigate a possible role for human rhinovirus c in respiratory exacerbations of children with cystic fibrosis, we conducted microbiologic testing on respiratory specimens from such patients in são paulo, brazil, during – . a significant association was found between the presence of human rhinovirus c and respiratory exacerbations. to investigate a possible role for human rhinovirus c in respiratory exacerbations of children with cystic fi brosis, we conducted microbiologic testing on respiratory specimens from such patients in são paulo, brazil, during - . a signifi cant association was found between the presence of human rhinovirus c and respiratory exacerbations. c ystic fi brosis (cf) is an autosomal inherited disease characterized by recurrent and chronic respiratory infections that ultimately lead to the need for a lung transplant early in life or to death ( ) . the role of bacterial infections in cf is well established, and most treatments focus on eradication or suppression of bacterial infections (mainly those caused by pseudomonas aeruginosa) ( ) . respiratory viruses such as respiratory syncytial virus (rsv) and infl uenza also seem to cause early damage or increase the risk for respiratory exacerbations ( , ) in these patients. however, the role of newly described respiratory viruses is not well known. infection of these patients with human rhinovirus (hrv), a member of the family picornaviridae, has been described. although some studies have suggested a substantial pathogenic role for these viruses ( ), controversy still exists ( ) . recently a new clade of human rhinovirus, named rhinovirus c ( ), was identifi ed through molecular methods. this new clade has been found throughout the world ( ) , and some studies have attributed severe respiratory infections in children to this agent ( , ) . we investigated whether this agent played a role in the respiratory exacerbations of children and adolescents with cf who attended the instituto da crinaça, hospital das clínicas da faculdade de medicina da universidade de são paulo. a total of cf patients ( girls, boys; median age . years; age range . months- . years) were enrolled in the study from september , through september , . nasopharyngeal aspirates or nasal mucus specimens for viral investigation, as well as sputum or oropharyngeal samples for microbiology culture, were collected during scheduled visits or unscheduled visits on occasions, with a median ± sd of ± . visits per patient (range - visits). clinical and lung function data were obtained at all visits. exacerbation of respiratory disease was defi ned as the presence of > of the following signs or symptoms: fever, increase in the amount of secretion or cough intensity, change in sputum's color, worsening of dyspnea, loss of appetite, a decrease of forced expiratory volume in s > %, and weight loss. total nucleic acids were extracted from nasopharyngeal samples by using a qiamp viral rna mini kit (qia-gen, hamburg, germany), according to manufacturer's instructions. reverse transcription was conducted with high capacity cdna archive kit (applied biosystems, foster city, ca, usa) by using μl of the previously extracted rna. respiratory viruses were identifi ed by individual reverse transcription-pcrs or pcrs selective for rsv; infl uenza viruses a and b; human parainfl uenza viruses , , and ; human coronavirus; human metapneumovirus; adenovirus; human bocavirus; picornavirus; and the β-actin gene ( ) . for picornavirus, we used the primer pair ol -ol , which include a portion of the ′ noncoding region (ncr) common to all picornaviruses, in the same conditions previously described ( ) . after sequence amplifi cation, products were examined by capillary electrophoresis in an automated dna sequencer (megabace, general electric healthcare-amersham biosciences, little chalfont, uk), and results were visualized through the megabace fragmentprofi ler software, which discriminates fragment sizes and fl uorescent intensities. samples in which picornavirus cdna had been identifi ed were submitted to a taqman-based real-time pcr protocol ( ) to identify hrv and enterovirus. only samples in which rhinovirus had been identifi ed by real-time pcr were submitted to ′ Νcr sequencing with an abi automated sequencer (applied biosystems), and results were submitted to the genbank database, accession nos. gu -gu . sequencing of the ′ ncr region has been shown to accurately discriminate among subtypes of rhinovirus, including genotypes c and a ( ) . all sequencing chromatograms obtained were edited manually to obtain contiguous fragments (contigs), by using sequence navigator software (applied biosystems). all sequences were screened at the national center for biotechnology information website by using the basic local alignment search tool (blast) (http://blast.ncbi.nlm.nih.gov/blast. cgi). hrv genotype was confi rmed by phylogenetic analysis as described below. sequences generated and a set of reference strains representative of hrv genotypes, available in the genbank database, were aligned by using clustalw (www.ebi.ac.uk/tools/clustalw ). minor manual adjustments were made to improve the alignment with bioedit software (www.mbio.ncsu.edu/bioedit/bioedit.html). the sequence of echovirus was used as the outgroup. phylogenetic analysis was performed with paup* version b (http://paup.csit.fsu.edu). neighbor-joining and maximumlikelihood trees were constructed on the basis of appropriate nucleotide substitution models determined by modeltest v . (university of vigo, vigo, spain). bootstrapping was assessed by using , replicates. trees were visualized by using the treeview program (www.taxonomy.zoology. gla.ac.uk/rod/treeview.html). to account for correlations among samples from the same patient, we used binomial generalized linear models to identify the virologic variables associated with the main endpoints (respiratory exacerbation and hospital admission). results were presented as odds ratios (ors) and % confi dence intervals. at least respiratory virus was identifi ed in ( . %) of samples; rhinovirus was the main identifi ed agent ( samples, . %). the results of virus identification, in relation to clinical status, are shown in table . co-infections were found in only samples ( . %); rhinovirus was the most frequent agent. sequencing was performed in of samples that were positive for rhinovirus and showed a predominance of genotype a ( samples; . %) (figure ). rhinovirus subtypes a and c were identifi ed in samples each. isolates in samples were identifi ed as hrv , coxsackievirus, and echovirus. therefore, isolates were enteroviruses, which indicates that the taqman-based real-time pcr platform misidentifi ed them. sequencing was not successful in sample. a maximum-likelihood phylogenetic tree of our samples and reference hrv sequences was constructed ( figure ). patients were examined during acute exacerbation of respiratory disease on occasions, and patients required hospital admission on occasions. the identifi cation of respiratory viruses was not associated with pulmonary exacerbations. because rhinovirus was the main agent identifi ed among stable patients, we verifi ed the effects of respiratory viruses, excluding rhinovirus from the analysis. a signifi cant association with respiratory exacerbation was found (or . , p = . ) ( table ). in contrast, when looking at rhinovirus subtypes, we noticed that identifi cation of rhinovirus subtypes a or c was also signifi cantly associated with respiratory exacerbations (or . ) ( table ). identifi cation of infl uenza a was the only variable associated with an increase in the risk for hospital admission (or . , % confi dence interval . - . , p = . ). these new hrv genotypes were initially described in samples from patients with infl uenza-like illnesses ( ) . recently, evidence has been increasing for the involvement of hrv-c in severe respiratory conditions such as bronchiolitis in infants ( ) and in exacerbation of asthma ( ) . however, these studies that attempted to clarify the patho-genicity of these new species did not include control groups or nonsymptomatic persons. in the study reported here, we obtained samples from patients during routine visits and exacerbations, which enabled us to identify a distinct role of different hrv subtypes. our fi ndings, however, cannot be extrapolated to infants with cf, which were underrepresented in our study. these infants may be at greater risk of hrv-c respiratory infections as they are for with rsv infections ( ) . previous studies of respiratory virus infections in cf patients provided confl icting results on the potential effect of rhinovirus. smyth et al. ( ) , who evaluated respiratory exacerbations in cf patients, identifi ed rhinovirus as the leading agent, with exacerbations following a more benign course but resulting in greater use of intravenous antimicrobial drugs ( ) . olesen et al. ( ) studied cf patients for year and used pcr to investigate the presence of different viruses in sputum or laryngeal aspirates. the group reported that viral infections did not reduce lung function or increase respiratory symptoms. rhinovirus was by far the leading agent identifi ed throughout their study ( ) . a more recent study by wat et al. ( ) , in which they used real-time nucleic acid sequence-based amplifi cation in conjunction with molecular markers to investigate the presence of respiratory viruses in children with cf, described an association of viral infections with respiratory exacerbations, particularly those caused by infl uenza a, infl uenza b, and rhinovirus ( ). we report infections by the novel rhinovirus subtypes a and c in cf patients. a signifi cant association was found between the presence of these agents and respiratory exacerbations. our fi ndings indicate the need for further investigation of hrv-c in cf patients. cystic fi brosis severe viral respiratory infections in infants with cystic fi brosis the role of respiratory viruses in cystic fi brosis viral and atypical bacterial infections in the outpatient pediatric cystic fi brosis clinic distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) global distribution of novel rhinovirus genotype clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children screening respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp -vp typing reveals high incidence and genetic diversity of hrv species c clinical and functional impact of acute viral respiratory infections in cystic fi brosis rhinoviruses replicate effectively at lower airway temperatures amplicon sequencing and improved detection of human rhinovirus in respiratory samples assay for ′ noncoding region analysis of all human rhinovirus prototype strains masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused infl uenzalike illness in new york state during erdman dd. novel human rhinoviruses and exacerbation of asthma in children effect of respiratory virus infections including rhinovirus on clinical status in cystic fi brosis this work was supported by fundação de amparo à pesquisa do estado de são paulo, grant / - .dr de almeida is a pediatric pulmonologist at instituto da criança hospital das clínicas da faculdade de medicina de universidade de são paulo and a phd candidate at the university of são paulo. her research interests are primarily related to respiratory diseases in children. key: cord- -fn m cn authors: philpott, erin k; englund, janet a; katz, joanne; tielsch, james; khatry, subarna; leclerq, stephen c; shrestha, laxman; kuypers, jane; magaret, amalia s; steinhoff, mark c; chu, helen y title: febrile rhinovirus illness during pregnancy is associated with low birth weight in nepal date: - - journal: open forum infect dis doi: . /ofid/ofx sha: doc_id: cord_uid: fn m cn background: adverse birth outcomes, including low birth weight (lbw), defined as < grams, small-for-gestational-age (sga), and prematurity, contribute to %– % of infant mortality worldwide and may be related to infections during pregnancy. the aim of this study was to assess whether febrile human rhinovirus (hrv) illness is associated with adverse birth outcomes. methods: active household-based weekly surveillance was performed for respiratory illness episodes in pregnant women as part of a community-based, prospective, randomized trial of maternal influenza immunization in rural nepal. rhinovirus (hrv) febrile illness episodes were defined as fever plus cough, sore throat, runny nose, and/or myalgia with hrv detected on mid-nasal swab. multivariate regression analysis evaluated the association between febrile hrv respiratory illness and adverse birth outcomes. results: overall, ( %) of pregnant women had hrv-positive febrile respiratory illnesses. infants born to pregnant women with hrv febrile illness had a . -fold increased risk of being lbw compared with those with non-hrv febrile illness ( of [ %] vs of [ %]; relative risk [rr], . ; % confidence interval [ci], . – . ). no difference in risk of lbw was observed between infants born to mothers with non-hrv febrile respiratory illness and those without respiratory illness during pregnancy ( of [ %] vs of [ %], respectively; rr, . ; % ci, . – . ). conclusions: febrile illness due to rhinovirus during pregnancy was associated with increased risk of lbw in a rural south asian population. interventions to reduce the burden of febrile respiratory illness due to rhinovirus during pregnancy may have a significant impact on lbw and subsequent infant mortality. adverse birth outcomes, including low birth weight (lbw), small-for-gestational age (sga), and preterm birth (ptb), contribute to %- % of infant mortality worldwide [ ] . infections during pregnancy impact risk of adverse birth outcomes and congenital anomalies [ , ] . respiratory viruses have been recognized as significant contributors to morbidity and mortality worldwide. influenza during pregnancy, in particular avian influenza and pandemic h n , has been associated with spontaneous abortion, stillbirth, and prematurity [ , ] . a prospective randomized trial in bangladesh and an observational us-based study each found an association between influenza immunization during pregnancy and a reduction in sga and an increase in mean birth weight [ , ] . an impact on lbw and ptb was additionally noted in a meta-analysis of randomized trials of maternal influenza immunization [ ] . human rhinovirus (hrv) is a respiratory virus first isolated in the s and historically implicated as the etiology of the common cold [ , ] . with improvements in molecular diagnostics, hrv has been increasingly recognized to cause serious illness requiring hospitalization and death among infants and young children, immunocompromised individuals, adults with chronic obstructive pulmonary disease, or asthma. human rhinovirus has also been identified as the most common etiology of pneumonia in hospitalized adults [ ] [ ] [ ] . however, the clinical presentation of hrv infection during pregnancy and effect on birth outcomes is not well established. the risk of increased susceptibility and severity associated with certain infections during pregnancy is likely related to a complex interplay of the maternal-placental-fetal unit that is not well understood. it has been postulated that infection causes activation of the innate immune system stimulating the release of proinflammatory cytokines, which may underlie the pathogenesis of both ptb and lbw [ ] . identification of a relationship between hrv infection during pregnancy and adverse birth outcomes has the potential to drive improvement of diagnostics, infection control measures, and treatment options for hrv infection. methods we conducted an analysis using samples and clinical data collected in a community-based, prospective placebo-controlled, randomized trial of maternal influenza immunization of pregnant women and their infants conducted in rural southern nepal from to . methods of the parent study have been previously published [ ] . in brief, illness episodes were identified through longitudinal household-based active weekly surveillance of women from the second trimester of pregnancy until months postpartum. mid-nasal swabs were collected from women meeting criteria for respiratory illness episode, which was defined as subjective fever plus or more symptom (cough, sore throat, runny nose, or myalgia). fever was included in the illness episode definition as part of the primary trial of maternal influenza immunization to capture influenza-like-illness during pregnancy [ ] . febrile hrv respiratory illness was defined as a respiratory illness episode with a nasal swab with hrv detected by real-time polymerase chain reaction (rt-pcr) assay. a unique illness episode was defined as an illness episode followed by symptom-free days. only the primary hrv illness episode was included in the analysis. adverse birth outcomes were defined as lbw (birth weight less than grams), ptb (birth at less than weeks gestation), and sga (birth weight < percentile of weight for gestational age). gestational age was calculated based on last menstrual period according to a house-to-house census conducted every weeks of all women of childbearing age [ ] . if a woman had a missed period, then a urine pregnancy test was performed and she was enrolled in the study if her urine pregnancy test was positive. therefore, the last menstrual period was used to date the infant's gestational age in the study with a -week recall period. the frequencies of each adverse outcome were compared between infants born to mothers with febrile hrv respiratory illness during pregnancy and those without febrile hrv respiratory illness during pregnancy. infant weights were included if they were obtained at less than hours after birth. small-for-gestational age was calculated based on the intergrowth- st criteria [ ] . a composite adverse birth outcome was defined as an infant being preterm, lbw, and/ or sga. real-time pcr for detection of hrv was performed as described, with the modification of a degenerate c/t base at the ' end of the forward primer [ ] . real-time pcr for additional respiratory viruses, including respiratory syncytial virus, human metapneumovirus, parainfluenza viruses - , influenza, coronavirus, bocavirus, and adenovirus, was performed using previously published methods [ ] [ ] [ ] [ ] [ ] . although the hrv rt-pcr assay has been shown to detect some enterovirus from culture lysates in which enterovirus was present in high copy numbers, this assay has not been positive when enterovirus-positive clinical respiratory specimens were tested as part of other studies and therefore less likely in this case to misdiagnose enterovirus as rhinovirus [ , ] . rhinoviruspositive samples collected from pregnant and postpartum women with cycle threshold values less than were selected for sequencing using a previously published protocol [ ] . for sequencing, previously extracted samples were used to generate complementary deoxyribonucleic acid using random hexamers, and sequencing was performed using primers targeting the ' noncoding region. gel-extracted amplicons were sequenced (genewiz) and analyzed using sequencher . software and compared with hrv reference sequences from genbank using the nucleotide-nucleotide basic local alignment search tool ([blast] www.blast.ncbi.nlm.nih.gov) algorithm. trimmed sequences were aligned using geneious r . (biomatters) and seaview . [ ] . phylogenetic reconstruction was performed using the maximum likelihood method with bootstrap replicates using phyml . within divein [ ] . neighbor joining trees were drawn and edited using figtree v . . . sequences were considered to be divergent genotypes if they differed from other study sequences by > % when compared using blast [ ] . sequence data were not of sufficient length to be submitted to genbank but are available upon request. log-linear regression was used to assess the association of baseline characteristics of pregnant women with the incidence of hrv respiratory illness during pregnancy; person-years (p-y) at risk of infection was included as an exposure. adverse birth outcomes such as lbw, sga, and ptb were also assessed using log-linear regression. potential risk factors analyzed include ( ) maternal febrile respiratory illness during pregnancy and, simultaneously, ( ) a specific history of hrv-associated respiratory illness. for the outcomes of lbw, we also included a term indicating hrvassociated respiratory illness in the second trimester, to assess whether timing during pregnancy was influential. to avoid confounding, additional covariates included season of birth and infant sex in the analysis. the wilcoxon-rank sum statistic was used to compare symptom duration between pregnant and postpartum women with febrile hrv as well as pregnant women who gave birth to lbw infants compared with those who did not. institutional review board (irb) approval for the parent study was obtained from the johns hopkins university bloomberg school of public health, seattle children's hospital, cincinnati children's hospital, the institute of medicine at tribhuvan university, and the nepal health research council. oral consent was obtained from study participants due to low literacy rates in the population. this procedure was approved by the irb. the trial in which this substudy was conducted is registered at clinicaltrials.gov (nct ). a total of ( %) of women had a febrile hrv respiratory illness episode, ( %) of which occurred during pregnancy and ( %) occurred after delivery. the median person-weeks (p-w) of follow up was weeks (interquartile range [iqr], - ) for febrile hrv respiratory illness during pregnancy and weeks (iqr, - ) for those without hrv-associated illness while pregnant. febrile hrv respiratory illness incidence was . / p-y. the incidence of febrile hrv illness while pregnant was of p-y, or . / p-y, and incidence after delivery was of p-y, or . / p-y (p = . ). characteristics of women with and without febrile hrv respiratory illness during pregnancy and their infants are outlined in table . the median gestational age at infection among the febrile hrv respiratory illness group was weeks (iqr, - ). incidence of hrv was similar by baseline characteristics, including maternal age, maternal literacy, nulliparity, indoor biomass cookstove use, and number of children in the household. in particular, no difference in maternal body mass index was observed. in addition, no difference was observed in influenza vaccination status between the groups. other viruses detected in association with febrile respiratory illness episodes included respiratory syncytial virus, human metapneumovirus, parainfluenza viruses - , influenza, coronavirus, bocavirus, and adenovirus each occurring at notably lower frequency compared with hrv. seventy-four of ( . %) infants born to pregnant women with febrile hrv respiratory illness and of ( . %) infants born to mothers without hrv-associated illness were weighed within hours of birth and therefore included in the assessment of lbw and sga. of infants with birth weight measured in the first hours, % ( ) were taken on the day of birth. median weight in those measured on days and of life was approximately . kg lower than those measured on day ( . at day and . at day vs . on day ), and the proportion determined to be of lbw increased from % on day to % on day to % on day . had all days observed exactly the same rate of lbw designations of %, it is expected then that approximately infants ( weighed on day and weighed on day of life) would not have been determined to have lbw. with infants classified as lbw, however, is only % of that group, so this comprises a very low potential misclassification rate. prevalence of lbw was significantly higher among infants born to pregnant women with febrile hrv respiratory illness compared with those born to mothers with a febrile respiratory illness of another viral etiology during pregnancy, after adjusting for season of birth and gender of the infant, with a . -fold increased risk of lbw ( (figure ; table ). when comparing pregnant women with non-hrv febrile viral respiratory illness to those . ) in the group of infants exposed to maternal febrile hrv illness compared with . kg ( . - . ) among infants not exposed to respiratory illness during pregnancy. three hrv infections occurred in the first trimester of pregnancy, in the second and in the third. the lack of hrv infection in the first trimester is due to enrollment of women into the study beginning at weeks gestation or later rather than lack of rhinovirus infection during the first trimester of pregnancy. low birth weight prevalence was not affected by the trimester of pregnancy in which the hrv infection occurred (p = . ). febrile hrv respiratory infection occurred yearround with a peak in october ( figure ) and accounted for a consistent % of respiratory virus infections each month. low birth weight occurred at a monthly rate of %- % (supplementary figure ) . due to the nature of enrollment, the number of patients under surveillance and therefore the numbers of samples collected were not constant over time, and this is reflected in the numbers of proportions of births and specimens tested (figure and supplementary figure ) . small-for-gestational age appeared higher among infants born to mothers with febrile hrv respiratory illness versus those with non-hrv febrile respiratory illness, although this did not reach statistical significance ( of [ . %] vs ( ) illness and symptom duration subdivided by lbw and pregnancy status are outlined in table . the median duration of illness was days (range, - ) among pregnant and postpartum women. no difference between overall illness duration was observed when comparing women with febrile hrv respiratory infection with and without lbw infants or when comparing women with febrile hrv respiratory illness during pregnancy versus after delivery. six women died during the period of surveillance, none of whom had febrile hrv respiratory infection. thirty-seven of ( %) women with febrile hrv respiratory illness during pregnancy sought medical care. overall, women sought care from a physician or a hospital. among women with febrile hrv respiratory infection during pregnancy, care-seeking behavior was similar between women who had lbw infants and those who did not ( of [ . %] vs of [ . %]; p = . ). in this same group with febrile hrv respiratory illness during pregnancy, the proportion seeking the care of a physician or hospital was also similar by lbw status of the infant. three of ( . %) who had lbw infants saw a physician, compared with of ( . %) who did not (p = . ). of the women with lbw infants who sought care from a physician, woman sought care for severe headache and fever, whereas the reason for seeking care from a physician for the other women was not documented. of successfully sequenced (supplementary figure ). of these, ( . %) were from pregnant women, whereas the remainder were from women who had rhinovirus after giving birth. species a ( of [ %]) accounted for a majority of the samples sequenced, whereas species b and c accounted for of ( . %) and of ( . %), respectively. among women who gave birth to lbw infants, of ( %) were species a and of ( %) were species b. however, the proportion of samples sequenced compared with the total was small. eleven ( %) pregnant women with hrv had an additional respiratory virus detected including coronavirus (n = ), human metapneumovirus (n = ), adenovirus (n = ), and bocavirus (n = ), and respiratory syncytial virus and bocavirus (each, n = ). coinfections occurred at equal frequencies among mothers with and without lbw infants ( of [ %] vs of [ %], respectively; p = . ). the association between lbw and febrile hrv-illness episode remained when infants born to mothers with febrile hrv with viral coinfection illness episodes were excluded from the analysis (rr, . ; % ci, . - . ; p = . ). no hrv-associated respiratory illness was found in association with influenza coinfection. over a -year period of prospective active home-based weekly surveillance in a rural subtropical setting, we characterize the incidence of febrile respiratory illness due to rhinovirus in pregnant women. limited data exist examining the relationship between hrv infection during pregnancy and adverse birth outcomes. in this study, we found that pregnant women with a febrile hrv respiratory illness were at . -fold increased risk to have a lbw infant, compared with pregnant women without hrv illness. several aspects of the study design allowed for an accurate assessment of the impact of febrile rhinovirus illness during pregnancy on adverse birth outcomes. the study was conducted in a region of the world where ptb and lbw are common. in addition, in a setting with limited antenatal care and frequent home births, birth weight was measured within hours of birth in the majority of infants. dating of gestational age was performed using last menstrual period obtained through -weekly surveillance for pregnancy in women of childbearing age, leading to more accurate dating of gestational age compared with recall of last menstrual period at time of birth. finally, intensive active weekly home-based respiratory illness surveillance allowed for collection of daily symptom data, and specimen collection in temperature-stable buffer permitted detection of rhinovirus and other respiratory viruses by sensitive molecular assays. similar to other subtropical regions, we found that febrile hrv respiratory infection occurred year-round with a peak in october and a nadir in june. low birth weight has been shown to vary by season due to factors such as food insecurity [ ] ; however, we observed a relatively steady rate of lbw among infants born to women without febrile hrv respiratory illness ranging from % to % without significant fluctuations. pregnant women are at high risk for severe complications due to influenza, with increased rates of morbidity, mortality, and adverse fetal outcomes most clearly demonstrated with pandemic h n [ ] . previous studies, including a study in our region, showed that febrile respiratory infection due to rsv was rare during pregnancy [ ] . limited data exist regarding the clinical presentation and severity of rhinovirus illness among pregnant women. human rhinovirus accounts for %- % of cases of viral pneumonia in hospitalized children and was detected in approximately half of children admitted to the intensive care unit for lower respiratory tract infection [ , ] . in adults, hrv is the most frequent cause of community-acquired pneumonia requiring hospitalization in the united states [ , , ] . we find that % of women with rhinovirus infection sought care for their illness, and that % of these were seen by a physician or at a hospital. this compares to an international study of community-based elderly adults, where % of those with rhinovirus illness were hospitalized [ ] . the relationship between febrile rhinovirus infection during pregnancy and lbw has not been well studied. low birth weight is the result of either preterm delivery or poor growth of the fetus resulting in intrauterine growth restriction (iugr). rates of ptb did not differ in women with and without febrile hrv respiratory illness during pregnancy, suggesting that iugr was the primary mechanism leading to lbw associated with hrv during pregnancy. we did not find a significant effect of febrile rhinovirus illness during pregnancy on ptbs or sgas. we note that approximately half ( %, or of ) of infants with any poor outcomes are affected by only of adverse birth outcomes (lbw, ptb, or sga); and only ( %) of infants are affected by all adverse birth outcomes. of infants with lbw, % are also sga (compared with % of who are not lbw but are sga). likewise, of with lbw, % are also preterm (compared with only % of who are not lbw but are preterm). therefore, although there is evident correlation between these outcomes, they are not completely overlapping. therefore, these outcomes are defining distinct populations and, in our study, may demonstrate different associations with risk factors. factors associated with iugr, including low socioeconomic status, literacy, parity, poor nutritional status, smoking, and alcohol use, were similar between the groups with and without rhinovirus in our study [ ] . nepal has one of the highest rates of malnutrition in south asia, with up to % of the general population having evidence of undernourishment [ ] . more importantly, in our study, pregnant women with and without hrv-associated respiratory illness had similar mean body mass indices of at the time of enrollment, suggesting that poor nutritional status associated with chronic illness is unlikely to be the etiology of the association with lbw. we also note that febrile illness during pregnancy alone was not associated with an increased risk of lbw in our study. of mothers with febrile non-hrv respiratory illness, only % of the infants were lbw, which is similar to the % without febrile respiratory illness during pregnancy. fever is associated with increased circulation of cytokines, in particular interleukin- , and has been postulated to be part of the reason why women with influenza may be at increased risk of severe disease and adverse birth outcomes [ ] . we did not find an effect of febrile respiratory illness without the detection of rhinovirus in this study, however, making this less likely to be responsible for the birth weight effect seen in this study. the birth weight effect in our study additionally does not appear to be due to increased duration or severity of illness in pregnant women with lbw infants. prolonged illness during pregnancy may be associated with decreased intake and poor weight gain. poor weight gain in pregnancy has been associated with iugr [ ] . we find that pregnant women with rhinovirus had a median days of symptoms, and that this did not differ in those with lbw or in those who had rhinovirus during pregnancy or after birth. no significant difference in hospitalizations, physician visits, or other care seeking was observed among pregnant women with febrile hrv illness who did and did not give birth to lbw infants. because of the potential for flu vaccination to reduce risk of lbw or ptbs, we evaluated for a difference in the incidence of rhinovirus febrile illness in women who did and did not receive the flu vaccine during pregnancy. we found no difference with flu vaccination. in addition, no mothers with rhinovirus febrile illness had evidence of influenza coinfection as a potential etiology of lbw. limitations of our study include our lack of active surveillance for respiratory viruses among asymptomatic or afebrile women, thereby limiting our ability to discern whether lbw was more strongly associated with hrv specifically or with any respiratory illness episode and its associated inflammation. use of subjective fever as criteria for respiratory illness rather than use of a measured temperature may have also decreased our ability to detect respiratory illnesses. we also did not conduct surveillance before weeks gestation, and, therefore, it is possible that some women had infections earlier in pregnancy that we were not able to identify in our study. gestational age dating was limited by lack of first-trimester ultrasound; however, -weekly prospective home surveillance minimized maternal recall error of last menstrual periods. illness episode severity measures, such as radiographic evidence of pneumonia or use of supplemental oxygen, were not evaluated because medical records of healthcare visits were not reviewed in this study. we were also unable to assess medical comorbidities in the participants, given the lack of access to primary or antenatal care in the study region. other limitations include lack of detection of bacterial pathogens in this study. human rhinovirus infections have been shown to augment respiratory bacterial colonization and infection; therefore, it is possible that bacterial infections may be contributing to the described febrile illnesses and play a role in the relationship with lbw. mid-nasal samples were only obtained once weekly, and brief episodes of viral shedding, as commonly observed in adults with pre-existing immunity, could have been missed. the association of virologic factors, such as viral load or subtype, with disease severity, were also unable to be assessed due to a lack of a method to quantify rhinovirus by our rt-pcr assay and our limited ability to sequence the majority of the isolates. the study was performed within a single geographic locale with specific demographics and risk factors, compromising generalizability. finally, because this is an observational study, we were unable to assess for a causal association between rhinovirus febrile respiratory illness and lbw. the groups with and without rhinovirus infection were similar in baseline sociodemographics, including factors that may affect lbw or risk of respiratory viral infections, such as maternal parity, maternal body-mass index, maternal literacy, household smoking, household density, and running water. in conclusion, we demonstrate that hrv is the most common cause of febrile respiratory illness in pregnant women in rural southern nepal during our study period, and that hrv during pregnancy was associated with an increased risk of lbw. because lbw infants have an increased risk of mortality compared with their heavier counterparts and hrv is a highly prevalent respiratory virus, this may represent a potential modifiable risk factor to reduce risk of lbw infants, particularly in developing countries. interventions to reduce the burden of febrile respiratory illness due to rhinovirus during pregnancy, such as measures to improve infection control, development of targeted therapies, or improvement of diagnostics, may have a significant impact on lbw and subsequent infant mortality worldwide. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. medimmune, and payments from glaxosmithkline for participation in a dsmb. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. global, regional, national, and selected subnational levels of stillbirths, neonatal, infant, and under- mortality, - : a systematic analysis for the global burden of disease study intrauterine infection and preterm delivery pregnancy and infection maternal and neonatal outcomes among pregnant women with pandemic influenza a(h n ) illness in florida, - : a population-based cohort study the effect of asian influenza on the outcome of pregnancy maternal influenza immunization and reduced likelihood of prematurity and small for gestational age births: a retrospective cohort study neonatal outcomes after influenza immunization during pregnancy: a randomized controlled trial the effects of influenza vaccination during pregnancy on birth outcomes: a systematic review and meta-analysis rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes viruses and bacteria in the etiology of the common cold human rhinoviruses community-acquired pneumonia requiring hospitalization among u.s. adults community-acquired pneumonia requiring hospitalization among u.s. children the preterm parturition syndrome designs of two randomized, community-based trials to assess the impact of influenza immunization during pregnancy on respiratory illness among pregnant women and their infants and reproductive outcomes in rural nepal impact of newborn skin-cleansing with chlorhexidine on neonatal mortality in southern nepal: a community-based, cluster-randomized trial international standards for fetal growth based on serial ultrasound measurements: the fetal growth longitudinal study of the intergrowth- st project real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses detection and quantification of human metapneumovirus in pediatric specimens by real-time rt-pcr comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children clinical disease in children associated with newly described coronavirus subtypes detection of bocavirus in saliva of children with and without respiratory illness molecular epidemiology of human rhinovirus infections in the pediatric emergency department rhinovirus disease in children seeking care in a tertiary pediatric emergency department seaview version : a multiplatform graphical user interface for sequence alignment and phylogenetic tree building divein: a web server to analyze phylogenies, sequence divergence, diversity, and informative sites a population-based prospective cohort emerging infections and pregnancy maternal effects of respiratory syncytial virus infection during pregnancy spectrum of respiratory viruses in children with community-acquired pneumonia lower respiratory infections among hospitalized children in new caledonia: a pilot study for the pneumonia etiology research for child health project respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness diagnosis and management of fetal growth restriction seasonal dietary intakes and socioeconomic status among women in the terai of nepal low maternal weight gain in the second or third trimester increases the risk for intrauterine growth retardation we thank the mothers and infants who participated in the trial and all the nepal nutrition intervention project-sarlahi staff involved in the study. key: cord- -x a sp authors: filntisis, panagiotis p.; zlatintsi, athanasia; efthymiou, niki; kalisperakis, emmanouil; karantinos, thomas; lazaridi, marina; smyrnis, nikolaos; maragos, petros title: identifying differences in physical activity and autonomic function patterns between psychotic patients and controls over a long period of continuous monitoring using wearable sensors date: - - journal: nan doi: nan sha: doc_id: cord_uid: x a sp digital phenotyping is a nascent multidisciplinary field that has the potential to revolutionize psychiatry and its clinical practice. in this paper, we present a rigorous statistical analysis of short-time features extracted from wearable data, during long-term continuous monitoring of patients with psychotic disorders and healthy control counterparts. our novel analysis identifies features that fluctuate significantly between the two groups, and offers insights on several factors that differentiate them, which could be leveraged in the future for relapse prevention and individualized assistance. wearable consumer products, such as smartwatches and fitness trackers, are gaining popularity every day and the enormous technological advances made in recent years have enabled reliable, unobtrusive and remote personalized collection of numerous behavioral and biometric signals through their sensors [ , ] . this so called "digital phenotyping" [ ] has enabled significant advances in wearables for health purposes, leading to the fact that next-generation wearable technologies are about to help transform nowadays hospital-centered healthcare practice to proactive, individualized care. behavioral and biometric indexes have been already used in general medicine and sports and nowadays the evidence indicates that they could be introduced into clinical psychiatry [ ] , as well. despite extensive research over the last years in neurobiology and neurophysiology of psychotic disorders, their cause remains unclear and reliable biometric indexes for the diagnosis and prediction of the course of the psychotic symptomatology have not yet been found. the use of such signals for the detection of early diagnosis and prevention of psychotic relapses is now one of the major research areas in psychiatry [ , , ] . the e-prevention project is an ongoing research and development project with the goal of collecting long-term continuous recordings of biometric and behavioral signals through non-intrusive commercial wearable sensors (i.e., smartwatches), in order to develop innovative, advanced and valuable tools. such tools would facilitate the effective monitoring, the prediction of clinical symptoms and the identification of biomarkers, which correlate with this research has been financed by the european regional development fund of the european union and greek national funds through the operational program competitiveness, entrepreneurship and innovation, under the call research-create-innovate (project code:t edk- ). more info can be found at: http://eprevention.gr behavioral changes in patients with psychosis so as to support the relapse prevention. timely detection of such relapses is in fact of major importance, not only for the clinicians; since patients not often present themselves when the symptoms begin to re-emerge or worsen [ ] , but it could also assist in reducing the severity of the relapses or even prevent their occurrences. in contrast with previous works, which have lasted from some hours to a few weeks [ , , ] , with some exceptions ( [ ] ), our ongoing research study has been going on for more than one year, with the goal of achieving two years of continuous monitoring. in addition, previous works have mostly used smartphones [ ] , and focused mainly in social features such as text messages, call duration or other such as location data, screen on/off time, and sleep duration. [ , , ] . compared to smartphones, wearable sensors are unobtrusive, lightweight and can be used for monitoring while the subjects perform daily activities [ ] , ensuring this way a safe and sound living environment. additionally, it has been already shown that people with psychotic illnesses are comfortable, able and willing to use personal digital devices to monitor outcomes in their daily life, supporting the fact that by using wearable sensors we could go beyond feasibility and underscore the novel physiological and activity data that can be easily collected with low cost [ , ] . in our work we employ a commercial off-the-shelf smartwatch, aimed to have minuscule intrusion in the subject's life and be worn / (except during charging). the nature of our long-term study asks for a different data processing approach than previous studies. inspired by traditional signal processing techniques, we extract common and more complex features using short-time analysis, and study them through their descriptive statistics in order to obtain a rough estimate of how they differentiate between healthy controls and patients with psychotic disorders. the experimental evaluation shows that both the more common, but also some of the novel nonlinear features examined are powerful in discriminating between the two groups. the analysis conducted in this work is a vital step towards developing a method that can leverage physiological and behavioral data from sensors in order to timely predict relapses or adverse drug reactions. table : demographics information of controls and patients at the time of recruitment, illness information, and amount of recorded data for each group during wakefulness and sleep. there were no significant differences for the recorded data. schizoaffective disorder) were recruited at the university mental health, neurosciences and precision medicine research institute "costas stefanis" (umhri) in athens, greece. all volunteers gave written consent for their participation after being fully informed about the project and also written permission for the use of their personal data (anonymized), in accordance with the provisions of the general regulation (eu) / . additionally, all protocols of the research project have been approved by the ethics committee of the institution. initially, the controls underwent a clinical evaluation to ensure there was no history of mental disorders or toxic substance usage, while for the recruitment of the patients, the clinicians met with the participants to conduct assessment of symptoms and functioning. at recruitment, patients were in active treatment and stable. the clinical team also conducted follow-up assessments with patients once every month of the study to administer various reliable rating scales (i.e., panss -positive and negative syndrome scale), which measure various psychiatric symptoms associated with their psychosis. table contains information on the demographics of the two groups as well as the collected data (described in sec. ) at the time of writing this paper. we also include the bmi (body-mass index) and the panss scale rating at the time of recruitment for the two groups (panss only applicable to patients). the subjects wore a samsung gear s smartwatch that continuously monitored acceleration (acc), angular velocity (gyr), and the heart rate (via photoplethysmography [ ] ). due to limits on the number of available devices, each subject was recruited at a different datecontrols were recruited between june and october while patients have been continuously recruited from november up to now (oct. ). controls were continuously monitored for at least days and then returned the watches, while the monitoring of patients is an ongoing process. in the analysis presented in this paper, to mitigate the effect of the covid pandemic quarantine lockdown ( / - / / in greece), we exclude data collected during this period. data were collected using an in-house developed application and uploaded every day to a secure cloud server [ ] . accelerometer (acc) and gyroscope data (gyr) were collected at a frequency of hz, while the heart rate and the heart rate variability (rr intervals -time intervals between two successive heart pulses) were collected at a rate of hz (if a new beat was not detected the watch duplicates the last obtained value). using the tizen api provided by the smartwatch, we also collected information about the sleep schedule of the subjects, and their steps at aggregated intervals of minutes. the heart rate variability (hrv) sequence from the hz signal was obtained by dropping identical consecutive values and ensuring that for each hour interval, the obtained sequence of rr intervals was summing up to at least minutes (an empirical threshold corresponding to % of valid heart data). we also removed rr intervals larger than ms and smaller than ms as artifacts and replaced possible non-detected pulses with linear interpolation. we did not perform any kind of noise reduction in acc and gyr, since we determined that for the examined features the effect of noise is negligible. short-time analysis of signals using windowing is a traditional signal processing method. in short-time analysis we assume the process under which the data are generated to be stationary. drawing power from these techniques, but largely increasing the time scale, we proceeded to perform "short-time" analysis in windows of minutes for movement data (acc, gyr) and hour for hrv. the minutes intervals that were chosen for the analysis of the movement data and the aggregation of the features have been found optimal for distinguishing short-term patterns in a previous study [ ] . the mean and standard deviation of the number of intervals for each user is reported in table . we consider the following features: energy the energy (ste) of the euclidean norm of acc and gyr is extracted (since they are measured triaxially). we use these features as an objective measure of physical activity and general movement behavior. spectral features medical studies split the hrv spectrum in four frequency bands: ultra-low-frequency (ulf ≤ . hz), very-low-frequency (vlf . - . hz), low-frequency (lf . - . hz), and high-frequency (hf . - . hz) [ ] . since hrv is by definition a non-uniformly sampled signal we perform spectral analysis using the lomb-scargle periodogram [ ] , and we extract for each interval the relative power and normalized power in two bands: lf and hf, as well as the ratio lf-to-hf. sample entropy nonlinear methods treat the extracted time series as the output of a nonlinear system. a typical characteristic of a nonlinear system is its complexity. the first measure of complexity we consider is the sample entropy (sampen). sample entropy is a measure of the rate of information generation by the system, considered an improvement over approximate entropy [ ] due to its unbiased nature. higuchi fractal dimension multiple algorithms have been proposed for measuring the fractal dimensions of a time series. here we use the higuchi fractal dimension [ ] , which has been used extensively in neurophysiology due to its simplicity and speed. multiscale fractal dimension (mfd) is an efficient algorithm [ ] that measures the short-time fractal dimension, based on the minkowski-bouligand dimension [ ] . real-world signals do not have the same structure over different time scales; and by measuring the mfd we are able to examine the complexity and fragmentation of the signals at multiple scales, thus creating a profile of local mfds at each time location. for this reason, we summarized the short-time measured mfd profiles by taking the following statistics: fd [ ] (the fractal dimension), min, max, mean, and std for the hour hrv data. poincare plot measures the poincare plot [ ] is a kind of recurrence plot where each sample of a time series is plotted against the previous, and then an ellipse is fitted on this scatter plot. the width of the ellipse (sd ) is a measure of short-term hrv, while the length (sd ) is a measure of long-term hrv. feature aggregation using the information on the sleep schedule of each subject we split the intervals in two groups -one corresponding to intervals during sleep and one during wakefulness. we then calculated the mean and standard deviation (std) over all its intervals, resulting in values for each subject and feature type; resulting in a total of features. sleep/wake ratio and steps in addition to the above features, we also extracted for each subject the mean and standard deviation of his sleep/wake ratio, and mean number of steps each day. since the number of recorded hours each day fluctuates, for these features only we keep data from subjects that have at least days with hours or more recorded ( controls with ± days and patients with ± days with no significant difference(p = . ). fig. shows examples of ste, and hrv fractal dimensions and lf/hf frequencies, during one day of monitoring a subject. in fig. (top row) we show the boxplots of the features during wakefulness that were deemed more fitting (due to space limitations) to display differences between the two groups. due to the differences observed perceptually between the distributions in most features, we tested for significant difference between distributions (the null hypothesis being that the two distributions are the same) using twotailed non-parametric mann-whitney u tests [ ] . we adjusted for p-values using the benjamini-hochberg (bh) procedure [ ] . due to the nature of our explorative study, bh was preferred over more strict family-wise error rate methods [ ] . table shows the results of mann-whitney u tests for all features, while the subjects are awake (due to limited space we omit the values of lf and hf normalized powers which showed no significant differences). during wakefulness, the features that pertain to movements appear to present more variability in the patient group when compared to controls. the same appears to be true for some nonlinear hrv features (sampen mean, higuchi, sd mean, sd , mfd max). the testing showed significant distribution differences in the standard deviation of acc and gyr short-time energy, the standard deviation of sd and sd , the sampen mean and mfd max and std. the other features failed to reject the null hypothesis. similarly, fig. (bottom row) presents the feature distributions for each group during sleeping. it is evident that especially the movement-related features present a significant difference, which is also verified in the mann-whitney u test results in table . a similar result is found for the standard deviation in sample entropy of hrv as well as the mean of the higuchi fractal dimension. mfd features (mfd fd [ ] std, max std, min std, and mean std) were also found to differ significantly. finally, fig. shows the boxplots of the statistics of steps per day and sleep wake ratio for the two groups. we observe a large significant difference between both the distributions of the mean and std of the sleep wake ratio (p < . ) as well as the distributions of total steps per day (p < . ). our goal with the statistical analysis in this work is to exploit traditional, but also less-known signal processing techniques to identify common markers/features that differ drastically when a person has a psychotic disorder. these markers could prove useful in predicting potential relapses in these patients. our findings have shown that patients tend to behave with greater variability and present large outliers -some behave close to controls, while others might show extreme values. during wakefulness, even though the mean energy did not differ when compared to controls, the standard deviation showed a significant difference, indicating that patients tend to depict large variations in their movement behavior. on the contrary, during sleeping the patients presented a small mean and standard deviation of the energy in each of their sleeping intervals compared to the controls. we should note however that the observed differences in sleep between the two groups could be attributed to medication administered to patients, which possibly causes variability in sleep duration as well. some of the nonlinear features that were measured for the hrv data showed significant differences in the distributions between controls and patients, i.e., during sleeping, as seen in table , such features are the standard deviation of the sample entropy, the mean of the higuchi fractal dimension, as well as various statistics derived from the mfd analysis (i.e., min, max, and mean of the standard deviation measurement). during wakefulness, the mean of the sample entropy, the std of the poincare features sd and sd and again various statistics of the mfd presented significant results. spectral analysis using the ls spectrogram did not show any significant differences between controls and patients. the main merits of our work are two-fold: first, compared to previous similar studies, which have mostly lasted for a few weeks, our study has already been going on for more than a year with the goal to obtain two years of continuous monitoring of patients with psychotic disorders. to do this, we employ a commercial off-theshelf smartwatch, that has been acknowledged by our volunteers to be comfortable and patients are willing to insert it into their daily lives routine. second, we show how traditional short-time-analysis combined with common but also more complex and novel features, such as the mfd features that depicted significant differences in both awake and sleeping data, can be employed to identify biomarkers, present large inter-group variabilities between healthy controls and patients, paving a way towards both acquiring clinical insights on psychotic disorders, but also exploring the capabilities of these markers to predict relapses. in this paper we identified markers that differentiate between healthy controls and people with psychotic disorders. to this end, we have specifically collected a large amount of physical activity and autonomic function data from wearable devices. statistical analysis between the two groups, through their descriptive statistics, indicated significant differences regarding the movement behavior, as well as in some markers of cardiac function during both wakefulness and sleeping. in future analyses, we also intend to account for the effects of antipsychotics and/or other medications administered to patients, as well as other factors that differ in the two samples, such as smoker/non-smokers percentages. finally, we aim to explore the capabilities of such markers to predict psychotic relapses and adverse drug effects. a review of wearable sensors and systems with application in rehabilitation the use of smartwatches for health monitoring in home-based dementia care new tools for new research in psychiatry: a scalable and customizable platform to empower data driven smartphone research sensing behavioral symptoms of mental health and delivering personalized interventions using mobile technologies rodromes and precursors: epidemiologic data for primary prevention of disorders with slow onset early recognition and disease prediction in the at-risk mental states for psychosis using neurocognitive pattern classification biomarkers and clinical staging in psychiatry from noncompliance to collaboration in the treatment of schizophrenia wearable monitoring for mood recognition in bipolar disorder based on historydependent long-term heart rate variability analysis relapse prediction in schizophrenia through digital phenotyping: a pilot study using wearable technology to detect the autonomic signature of illness severity in schizophrenia predicting early warning signs of psychotic relapse from passive sensing data: an approach using encoder-decoder neural networks human activity recognition on smartphones with awareness of basic activities and postural transitions crosscheck: integrating self-report, behavioral sensing, and smartphone use to identify digital indicators of psychotic relapse wearable sensors for human activity monitoring: a review do we still have a digital divide in mental health? a five-year survey follow-up a comparison of passive and active estimates of sleep in a cohort with schizophrenia photoplethysmography and its application in clinical physiological measurement an intelligent cloud-based platform for effective monitoring of patients with psychotic disorders person identification using deep convolutional neural networks on short-term signals from wearable sensors an overview of heart rate variability metrics and norms studies in astronomical time series analysis. iistatistical aspects of spectral analysis of unevenly spaced data physiological timeseries analysis using approximate entropy and sample entropy approach to an irregular time series on the basis of the fractal theory fractal signal analysis using mathematical morphology fractal geometry: mathematical foundations and applications do existing measures of poincare plot geometry reflect nonlinear features of heart rate variability? on a test of whether one of two random variables is stochastically larger than the other controlling the false discovery rate: a practical and powerful approach to multiple testing a general introduction to adjustment for multiple comparisons key: cord- - qgaxcqq authors: scott, e. m.; magaret, a.; kuypers, j.; tielsch, j. m.; katz, j.; khatry, s. k.; stewart, l.; shrestha, l.; leclerq, s. c.; englund, j. a.; chu, h. y. title: risk factors and patterns of household clusters of respiratory viruses in rural nepal date: - - journal: epidemiol infect doi: . /s sha: doc_id: cord_uid: qgaxcqq viral pneumonia is an important cause of death and morbidity among infants worldwide. transmission of non-influenza respiratory viruses in households can inform preventative interventions and has not been well-characterised in south asia. from april to april , household members of pregnant women enrolled in a randomised trial of influenza vaccine in rural nepal were surveyed weekly for respiratory illness until days after birth. nasal swabs were tested by polymerase chain reaction for respiratory viruses in symptomatic individuals. a transmission event was defined as a secondary case of the same virus within days of initial infection within a household. from households, initial viral illness episodes occurred, resulting in transmission events. the overall incidence of transmission was . events per person-weeks. risk of transmission incidence was associated with an index case age – years (incidence rate ratio (irr) . ; % confidence interval (ci) . – . ), coinfection as initial infection (irr . ; % ci . – . ) and no electricity in household (irr . ; % ci . – . ). preventive interventions targeting preschool-age children in households in resource-limited settings may decrease the risk of transmission to vulnerable household members, such as young infants. acute lower respiratory infection (alri) is the primary cause of child morbidity and mortality worldwide with the vast majority of childhood deaths related to alri occurring in resourcelimited settings [ ] . respiratory viruses are increasingly recognised as a cause of severe alri in young children [ ] . in many global regions where access to healthcare is limited, especially in rural areas, the true community-based burden of respiratory virus-associated alri remains poorly characterised [ ] [ ] [ ] . in these settings, household surveillance studies can provide a more comprehensive evaluation of viral incidence, transmission and molecular epidemiology patterns in the community [ ] [ ] [ ] [ ] [ ] . household surveillance can provide valuable information regarding the transmission networks within households. such knowledge may guide the development and implementation of preventative interventions to protect vulnerable groups from alri. for example, infants are at highest risk for severe alri from respiratory syncytial virus (rsv) [ ] . major challenges to developing a safe and effective rsv vaccine in young infants have resulted in the development of alternative strategies including maternal rsv vaccination and delayed vaccine administration until > months of age [ ] . targeting older groups for vaccination may protect vulnerable populations by interfering in transmission chains to young infants, the elderly and other high-risk groups. studies in rural kenya have identified school-age children as the primary introducers of rsv into households where an infant subsequently became infected [ ] . these results were in agreement with a us study from the s reporting older siblings aged - years as most likely to introduce rsv disease into families [ ] . in contrast, modelling suggests that young children < years are more likely to transmit rsv and are the most efficient population to vaccinate in order to prevent disease in other groups [ ] . few studies have analysed the transmission of other non-influenza respiratory viruses, such as human metapneumovirus (mpv) and human rhinovirus (hrv), and no studies have examined the household transmission dynamics of respiratory viruses in rural south asia, a region characterised by high rates of preterm birth and infant mortality [ , [ ] [ ] [ ] . the aims of this analysis were to characterise the transmission of nine non-influenza respiratory viruses within households in rural nepal and to determine household characteristics associated with the transmission of respiratory viruses. we hypothesise that the presence of school-age children in a household will be associated with an increased risk of transmission. this prospective household surveillance study was nested within a randomised controlled trial designed to determine the effectiveness of influenza vaccine during pregnancy [ , ] . the study site is in the low-lying region of rural southern nepal called the 'terai', with inhabitants broadly representative of the population of india, bangladesh and nepal [ ] . all women of childbearing age in a part of one district were surveyed for pregnancy. pregnant women were enrolled in the primary trial as early as possible in pregnancy, generally during the second trimester, and followed until months postpartum. at the time of randomisation, every third study mother and their families were selected to participate in a household surveillance substudy. as randomisation occurred at the time of vaccination, not initial enrolment, randomisation occurred after enrolment and the start of surveillance for some mothers. surveillance for the first participant enrolled in the household substudy began on april and we included the households of substudy mothers enrolled prior to may . surveillance of the household ended days after birth. in this area, many households consist of multiple families living in a single compound; households were defined as a group sharing a single cookstove. socio-demographic data were collected upon enrolment at the individual and household levels. birth assessments of study infants were performed shortly following birth. infants weighed within h of the birth were considered to be low birthweight if the infant weighed < . kg. trained field staff visited the home weekly and used a standardised form to inquire about respiratory symptoms and signs in mothers, infants and other household members for each day in the previous week. a mid-nasal turbinate swab was collected from mothers and other adult household members aged ⩾ years with self-reported fever, plus one or more of the following symptoms within the previous days: cough, sore throat, runny nose, nasal congestion or myalgias. swabs were collected from all children < years with at least one of the following symptoms: subjective fever, cough, draining ear, wheezing or difficulty breathing, in the previous days. illness episodes were defined as symptoms that met described criteria and were separated by at least seven symptom-free days. only individuals with ⩾ days of symptom diary recorded, with or without illness, were included in the analyses. households that did not have ⩾ individuals with surveillance were excluded from the analysis as two-person households consisted of mother-infant pairs without surveillance of household members. respiratory swabs were collected, aliquoted and transported from the nepal field site to the university of washington in seattle, wa in a temperature-stable buffer (primestore; longhorn diagnostics, san antonio, usa). samples were tested by a real-time reverse transcriptase polymerase chain reaction (pcr) for respiratory viruses, including rsv, mpv, influenza viruses a and b, parainfluenza virus - (piv - ), adenovirus (adv), human coronavirus (cov), hrv and bocavirus [ ] [ ] [ ] . influenza transmission in household was the primary aim of the trial substudy and is being analysed separately. bocavirus was not included because of its prolonged shedding patterns. sequencing was performed for hrv-and rsv-positive samples from household illness clusters utilizing samples with pcr cycle threshold values < and for hrv and rsv, respectively, based on previous difficulty sequencing low viral load samples [ , ] . briefly, nucleic acid was extracted, and cdna was synthesised. a hemi-nested pcr protocol was used targeting the ′ untranslated region and the second hypervariable region of the attachment (g) glycoprotein coding region for hrv and rsv, respectively [ , ] . sequences were aligned using mafft v . , and maximum likelihood phylogenetic trees using the hky model with bootstrap replicates were inferred using phyml . within geneious [ , ] . sequences were considered to be the same virus type with ⩾ % identity. sequences > base pairs (bp) were submitted to genbank under accession numbers mh to mh . for each examination of viral transmission, initial viral infections were those preceded by a -day period without infections of that virus type in a household. index or transmitting cases were established by identifying the individual(s) with the earliest reported respiratory symptoms prior to a virus-positive swab. we computed the incidence of secondary household illness cases following each initial viral infection. we defined transmission events as observed infections of the same virus in another member of the same household within the subsequent days. the -day risk period began on the first day of criteria respiratory symptoms associated with virus-positive specimen collection in the week preceding the virus-positive swab in the index case. each initial infection and its corresponding risk period comprise a single data point in the regressions and include subsequent infections and time at risk from all household members reporting symptoms over that time period. to assess the risk factors for the incidence of secondary cases of these initial illnesses within households, we used generalised estimating equations, accounting for the potential similarity among repeated initial infections within households. the outcome was the number of secondary cases, with a fixed offset of the log time at risk; a log link was used to estimate the incidence rate ratios (irrs). multivariable regression was performed by first including all measures significant in the univariable analysis at p < . and then performing backward elimination to select a final model. measures were retained in final multivariable regression if significant at p < . or if had a substantial impact (⩾ % shift in estimate) on other significant covariates. a single index case type was included in the multivariable model as the index case was coded such that one index case type was compared to all other index cases. potential risk factors for transmission included maternal and household characteristics. some households included more than one enrolled mother-infant pair. among households with more than two mothers, household characteristics were compared with a sensitivity analysis using one mother's descriptors vs. the others. data were analysed using sas/stat . (sas institute inc.) and stata (stata corp) statistical software. institutional review board approval for the randomised controlled trial was given by the johns hopkins university bloomberg a total of households were enrolled with a median household size of (range - ). five-hundred and fifty-five households contributed symptom reporting from at least three persons. within the surveyed households, out of ( %) initially enrolled household members were surveyed for weekly respiratory illness. these individuals included in the transmission analysis consisted of mothers, infants, other adults ⩾ years and other children < years ( fig. ) . characteristics of all households and individual characteristics of surveyed individuals are summarised in table . within included households, % of study mothers and infants were surveyed, whereas only % of other adult household members and % of other children were surveyed. the proportion of other adults with surveillance was % vs. % among individuals < vs. ⩾ years, and % vs. % in males compared to females. forty-nine per cent of other children aged - years were surveyed compared to % of other children aged < years. of children aged - years attending school, % were surveyed compared to % of non-school attending children. a total of virus-positive initial illness episodes occurred within households with a median of one (range - ) illness episode per household. in the days following initial household illness, subsequent illness episodes occurred, ( %) of which were screened by pcr and ( %) illnesses that did not have a swab collected despite meeting symptom criteria. eight per cent of illness episodes resulted in a pcr-confirmed secondary case within the household with a total of transmission events in household illness episodes. household illness clusters occurred in households as some households experienced multiple illness clusters. the incidence of a pcr-confirmed transmission event of any virus occurring in the days following initial fig. ). hrv coinfection with another respiratory virus had more frequent transmissions ( . % of hrv coinfections resulted in transmission) compared to monoinfection of hrv ( . %), coronavirus ( . %) and rsv ( . %) (fig. , see table for statistical testing). we evaluated risk factors for transmission following initial respiratory viral illness ( fig. s ). of nine fully evaluated hrv transmission events, household sequences matched in six ( . % of all hrv transmission events). in three events ( . % of all transmission events), the individuals were infected with different hrv genotypes (fig. ). all other episodes had insufficient data to confirm the transmission of specific viral genotypes. of households contributing to the regression analysis of any virus transmission, households included more than one mother-infant pair. within households with multiple mothers, there was % agreement in reporting of electricity within the home, % agreement in indoor cookstove use and % agreement of latrine within the home. the multivariable regression model was performed using the alternative mother's household information with similar results (supplementary table s ). a sensitivity analysis was also performed using a definition of transmission as a secondary case of the same virus within days of initial infection within the household. using this definition, preschool child index case, coinfection as initial infection and a low birthweight infant in the household were associated with an increased incidence of household transmission (supplementary tables s and s ). in a prospective longitudinal study utilizing intensive weekly home-based active surveillance to evaluate the household transmission of nine respiratory viruses in rural south asia, initial infection in young children was associated with the greatest risk of symptomatic respiratory virus household transmission with spread to infants occurring in % of transmission events. southern nepal is a region where household crowding is common and there are high rates of infants born prematurely or low birthweight [ , ] . our data demonstrate a significant burden of symptomatic respiratory viral illness in households; based on a multivariable model, young children and socio-cultural factors, such as socio-economic status, may predispose to the transmission of viruses in this region. in over % of transmission events of all viruses, preschool children (aged - years) served as an index case. a higher proportion of initial infection among this group resulted in secondary cases compared to other age groups, including school-age children and mothers, a finding confirmed in our multivariable model of transmission incidence. in rsv transmission, no index cases were older children and % of index cases were mothers. in contrast to our findings, a study of rsv transmission in the usa during the s found that older siblings between and years most frequently introduced rsv into a household [ ] . similarly, a kenyan household study examined sequencingconfirmed rsv transmission in households with infants and identified school-age children as the most common index case resulting in infant infection [ ] . our finding that preschool-age children, rather than schoolage children, are most likely to transmit non-influenza respiratory viruses is likely due to differences in study sample and design, as well as transmission patterns. households in our study experienced fewer respiratory viral illness episodes than reported in other household studies that included asymptomatic viral detections [ , , ] . in a study of respiratory virus-positive influenzalike illness in households in vietnam, households experienced . illness episodes over a -year period (including influenza and bocavirus), whereas we found a mean of . illness episodes per household [ ] . our surveillance sample includes a higher proportion of young children aged - years, including study infants, relative to the overall proportion in the sarlahi district of nepal ( . % vs. . %) and a lower proportion of children aged - years ( . % vs. . %) [ ] . it is possible that the true transmitting cases were absent during the weekly household visit or asymptomatic according to our criteria, though this is less likely for younger children who have median viral shedding duration of longer than week [ ] . we likely did not capture the full a total for initial episodes, transmission episodes and transmission events are less than the sum of columns as infections with coinfections were counted a single initial infection in total. similarly, the total for index cases may be less than the sum of columns as coinfection transmission or transmission to multiple household members was only counted once in total. b median serial index was defined as the median number of days between symptom onset of index and secondary case. contribution of older children to transmission compared to the kenyan cohort. over half of rsv infections in children - years in that study were asymptomatic, with a smaller proportion of asymptomatic infections in infants under year and children - years at % and %, respectively [ ] . however, they also reported viral shedding in symptomatic rsv infections was log rna copies greater than in asymptomatic rsv cases suggesting that symptomatic episodes are more likely to transmit virus [ ] . last, we collected weekly specimens and our findings may be biased if non-infant younger children had longer shedding duration compared to older children. however, this has not been demonstrated in studies of rsv and hrv shedding duration [ , ] . our large cohort allowed us to use a multivariable analysis to identify the risk factors and protective characteristics associated with the incidence of transmission. while both infants and preschool children were frequently identified as the index case in a transmission event and can shed virus for prolonged periods, a preschool child index case was associated with a twofold increased risk of transmission and an infant index case was associated with a decreased risk of transmission [ , , ] . whereas infants are more likely to transmit rsv via direct contact as compared with fomites, young children may transmit infection efficiently through both methods due to differences in mobility and behaviour [ ] . coinfection as the initial infection was associated with an increased risk of transmission, including in our multivariable model. coinfections most commonly involved hrv and a greater proportion of coinfections resulted in a secondary case compared to monoinfection of most viruses. viral coinfection with rsv infection has been demonstrated to increase rsv viral load and shedding duration. however, this has not been consistently seen, including a study analysing seven respiratory viruses [ , ] . finally, electricity in the household, a proxy for socio-economic status and housing conditions, was negatively associated with transmission. although an association between indoor air pollution and rsv infection has been reported in resource-limited regions, smoking and biofuel cookstove use were not associated with the risk for transmission in our model [ ] . however, we had limited power to detect this association due to the use of indoor biofuel cookstoves in over % of households in our model and exposures were self-reported without actual measures of indoor air pollution. a study in peru demonstrated that age, occupation and household size can influence contact network size and pattern [ ] . our findings, from a population consisting of crowded households, lower levels of maternal education and fewer children attending school compared to other household transmission studies, suggest that differences in socio-demographic, cultural and environmental contexts influence household transmission risk factors, including the source of household introduction. as we actively surveyed all women of childbearing age for pregnancy, our cohort is generalisable to households with young infants in southern nepal, a region representative of rural south asia [ ] . in the sarlahi district during the study period, an estimated % of the population were below the poverty line and approximately % of infants were born low birthweight and % preterm [ , ] . households in our study were crowded with over one-third containing > people per room and multiple family units frequently living in a single structure. the average population is young; the median age in the sarlahi district was years [ ] . eighty-four per cent of households used indoor biofuel cookstoves and half contained latrines. young demographics, crowded housing conditions and socio-economic factors may influence the patterns of respiratory (a and b) . each row represents an individual, each unfilled symbol represents day of symptoms, black filled symbols represent positive specimen collection and varying symbols represent household member type. index cases are those whose symptoms first appear before the initial rsv-positive specimen. [ ] . this socio-demographic, environmental and cultural context should be considered when implementing preventative strategies for the control of respiratory viral illness, such as vaccines, antivirals, hygienic measures and physical barriers. for example, there are multiple rsv vaccines targeting diverse populations from infants and children to pregnant women and other adults in various stages of clinical trials [ ] . because the immune systems of neonates generally do not respond well to primary vaccination, immunizing mothers and other household members has been proposed as a method to protect vulnerable young infants from rsv [ ] . while a model of kenya transmission data supports immunizing school-age children to diminish transmission of the virus to infants, our study suggests that in rural south asia, preschool-age children are more likely to transmit respiratory viruses to other household members [ ] . this suggests that a 'one-size fits all' approach to rsv vaccine implementation, or other respiratory viral transmission prevention measures, may not be effective as transmission dynamics may differ across global settings. our study has several limitations. asymptomatic infections were not captured, affecting our ability to fully characterise the transmission chain. we expect that asymptomatic transmission may have impacted our ability to characterise hrv spread, particularly in transmission involving older children and adults, our ability to associate age of index case with transmission risk [ , ] . moreover, we likely only captured a minority of adult illness as adults required subjective fever for specimen collection and fever occurs infrequently in adult rsv, mpv and hrv illness [ ]. while we underestimated transmission, specifically spread involving individuals ⩾ years, due to our symptom criteria, a previous study of rsv transmission demonstrated that the odds of transmission in symptomatic infection is five times that of asymptomatic infections [ ] . this suggests that we likely captured index infections. we anticipate that some illness with shedding < days may have been missed due to our weekly surveillance. we expect that this represented a small minority of illness episodes as the estimated shedding duration of hrv and rsv in adults is and days, respectively [ , ] . shedding for - weeks is common with paediatric respiratory infections [ ] . moreover, while we performed sequencing on rsv and hrv samples involved in transmission chains, we were not able to phylogenetically verify transmission in the majority of episodes due to high cycle threshold values, and could not use sequencing data to define transmission. our sequencing results revealed a degree of misclassification with some hrv and rsv transmission events representing illness clusters with multiple virus types circulating in the household simultaneously. this was especially true for hrv, a finding in agreement with previous studies of hrv transmission, including in household and daycare settings [ , ] . additionally, some households originally selected for the household substudy were not surveyed as intended. individuals within selected households who were not surveyed were primarily adults. among adults, males and those < years old were surveyed less frequently, a group not considered high risk for household transmission of respiratory viruses in previous studies. a significant proportion of the sarlahi population, especially men, are reported as absent from home, supporting the possibility that some household members were absent from the community during the study, although these data were not captured [ ] . we also surveyed a higher proportion of preschool children compared to school-aged children. the differential exclusion of these subsets may have affected our results, including identification of index cases, especially if these persons were periodically present in the household. lastly, we did not collect data on the social mixing patterns of individuals in these households which would have provided valuable information regarding possible causal explanations for our findings. these results provide data that may help to optimise the implementation of preventative strategies with the aim of protecting vulnerable infants. south asia is an area of the world with a high incidence of low birthweight infants, household crowding and malnutrition, all risk factors for severe childhood alri. our study of non-influenza respiratory virus transmission within households in rural nepal highlights the importance of targeting preschool-age children to prevent the spread of respiratory viral illness. understanding the household transmission of respiratory viruses in rural resource-limited populations will help evaluate infection prevention strategies, such as immunisation of mothers and other household members in protecting infants, who are most vulnerable to respiratory viral infection. global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in : a systematic analysis viral pneumonia a prospective three-year cohort study of the epidemiology and virology of acute respiratory infections of children in rural india incidence and risk factors for respiratory syncytial virus and human metapneumovirus infections among children in the remote highlands of peru continuous invasion by respiratory viruses observed in rural households during a respiratory syncytial virus seasonal outbreak in coastal kenya the source of respiratory syncytial virus infection in infants: a household cohort study in rural kenya community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study epidemiology and etiology of influenza-like-illness in households in vietnam; it's not all about the kids! the burden of respiratory syncytial virus infection in young children strategic priorities for respiratory syncytial virus (rsv) vaccine development respiratory syncytial virus infections within families vaccination strategies against respiratory syncytial virus factors associated with transmission of influenzalike illness in a cohort of households containing multiple children household transmission of respiratory virusesassessment of viral, individual and household characteristics in a population study of healthy australian adults rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections designs of two randomized, community-based trials to assess the impact of influenza immunization during pregnancy on respiratory illness among pregnant women and their infants and reproductive outcomes in rural nepal year-round influenza immunisation during pregnancy in nepal: a phase , randomised, placebo-controlled trial national population and housing census evaluation of quantitative and type-specific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children molecular epidemiology of human rhinovirus infections in the pediatric emergency department molecular epidemiology of respiratory syncytial virus transmission in childcare new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . recent developments in the mafft multiple sequence alignment program mortality risk in preterm and small-for-gestational-age infants in low-income and middle-income countries: a pooled country analysis nepal human development report frequent asymptomatic respiratory syncytial virus infections during an epidemic in a rural kenyan household cohort quantification and determinants of the amount of respiratory syncytial virus (rsv) shed using real time pcr data from a longitudinal household study virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia influence of age, severity of infection, and co-infection on the duration of respiratory syncytial virus (rsv) shedding epidemiology of multiple respiratory viruses in childcare attendees modes of transmission of respiratory syncytial virus multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children a household-based study of contact networks relevant for the spread of infectious diseases in the highlands of peru nepal living standards survey / : statistical report maternal immunization evaluating vaccination strategies for reducing infant respiratory syncytial virus infection in low-income settings an intensive, active surveillance reveals continuous invasion and high diversity of rhinovirus in households the presence of fever in adults with influenza and other viral respiratory infections heterotypic infection and spread of rhinovirus a, b, and c among child care attendees acknowledgements. we thankfully acknowledge the original principal investigator of the main study, the late mark c. steinhoff. we acknowledge the contribution of the nepal nutrition intervention project study team in sarlahi and kathmandu. supplementary material. the supplementary material for this article can be found at https://doi.org/ . /s . key: cord- -y z w x authors: nan title: copd sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: y z w x nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- - h msv authors: kistler, amy; avila, pedro c.; rouskin, silvi; wang, david; ward, theresa; yagi, shigeo; schnurr, david; ganem, don; derisi, joseph l.; boushey, homer a. title: pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: h msv background. between % and % of asthma exacerbations are associated with viral respiratory tract infections (rtis), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood because of the limited scope and throughput of conventional viral detection methods. methods. we investigated the capability of the virochip, a dna microarray—based viral detection platform, to characterize viral diversity in rtis in adults with and without asthma. results. the virochip detected viruses in a higher proportion of samples ( %) than did culture isolation ( %) while exhibiting high concordance ( %) with and comparable sensitivity ( %) and specificity ( %) to pathogen-specific polymerase chain reaction. a similar spectrum of viruses was identified in the rtis of each patient subgroup; however, unexpected diversity among human coronaviruses (hcovs) and human rhinoviruses (hrvs) was revealed. all but one of the hcovs corresponded to the newly recognized hcov-nl and hcov-hku viruses, and > different serotypes of hrvs were detected, including a set of divergent isolates that formed a distinct genetic subgroup. conclusions. the virochip can detect both known and novel variants of viral pathogens present in rtis. given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation. tory syncytial virus (rsv) (reviewed in [ ] ). in older children [ ] and adults [ , ] , human rhinoviruses (hrvs) are implicated in the majority of cases, with variable contributions from human coronaviruses (hcovs), influenza viruses, parainfluenza viruses, rsv, and human metapneumovirus (hmpv) (reviewed in [ ] ). however, the factors that determine the clinical outcomes associated with rtis in persons with asthma are not well understood. host factors governing inflammatory and immune responses have been demonstrated to influence whether a host with asthma experiencing a viral rti will develop an exacerbation of symptoms [ ] [ ] [ ] [ ] . in contrast, it remains unresolved whether variation in viral pathogens may also influence this outcome. for example, despite the prominent association between hrvs and asthma exacerbation, deliberate inoculation of a laboratory-adapted hrv- isolate into subjects with asthma did not produce wheezing, despite efficient infection and the generation of upper respiratory tract symptoms [ ] [ ] [ ] [ ] [ ] [ ] . although these experimental hrv inoculation studies have not demonstrated clinically significant exacerbations, a small decline in pulmonary function has been observed [ , ] . likewise, not every cold in subjects with asthma is associated with exacerbation of asthma symptoms. these findings raise the possibility that some respiratory viral pathogens may have greater potential than others of triggering asthma exacerbations in susceptible hosts. if this is so, then the spectrum of viral isolates recovered from rtis in persons with asthma who experience exacerbations should differ from that in persons with asthma whose rtis do not trigger exacerbations. few prior studies have attempted to make this comparison, and those that did have generally not used methods that can differentiate among the different subtypes of viruses within a family. estimating the number of hrv isolates, rather than identifying them by serotype or genotype, may be insufficient given that certain isolates of hrv are demonstrably inefficient at triggering asthma on inoculation. part of the reason for the paucity of prior studies of this type derives from the limitations of conventional viral detection methods. viral culture is insensitive, and antigen testing, while reasonably sensitive, is ( ) unavailable for several important classes of pathogen, notably hrvs and hcovs, and ( ) not designed to discriminate among isolates of a given viral species. polymerase chain reaction (pcr) methods have high sensitivity but are limited to the detection of previously characterized or highly conserved viral sequences. we have developed an alternative, comprehensive strategy for viral detection that uses the virochip, a dna microarray bearing the most conserved sequences of all known viruses of humans, animals, plants, and microbes [ , ] . in addition to the ability of the virochip to detect known viruses, it can also detect new members of known virus families by crosshybridization-a significant advantage over pcr-based methods [ ] [ ] [ ] [ ] . here, we have applied the virochip to an ongoing prospective study designed to analyze the diversity of viruses that cause rtis in adults with and without asthma, with particular attention given to those that provoke asthma attacks. we recruited adults with and without asthma by advertising on a community web site (craigslist; http://sfbay.craigslist.org/), by posting flyers on campus, and by sending e-mail messages to subjects who had responded to previous advertisements for research studies. participant recruitment started in the fall of and extended through the end of december . the announcements requested that subjects contact us within h of the onset of "cold" symptoms. we evaluated subjects first within - days of cold onset (visit ), then again between days and of cold symptoms (visit ), and at weeks or longer thereafter to assess baseline status (visit ). eighty-three subjects were enrolled; had asthma, and did not. asthma was defined as a history of asthma symptoms (recurrent dyspnea, wheezing, and chest tightness) associated with a positive methacholine test result [ ] . subjects without asthma had neither a history of asthma symptoms nor a positive methacholine test result. all were informed of the purposes, procedures, and risks of participating, and all signed informed-consent forms approved by the university of california, san francisco, committee of human research. study procedures. at the first visit, participants completed questionnaires on demographics, medical history, asthma history, and date of onset of cold symptoms. at each visit, participants provided information on the severity of current upper and lower respiratory tract symptoms by use of a validated cold questionnaire [ ] and diary form [ ] that have been described elsewhere. spirometry measurements (forced expired volume in s [fev ] and forced vital capacity [fvc]) were obtained, and the percentage predicted was calculated using crapo equations. nasal lavage (nl) was then performed by instilling ml of warmed normal saline into each nostril and, after a -s dwell time, having the subject expel the nasal contents into a plastic cup. subjects with asthma recorded symptom severity and a.m. and p.m. peak flow measurements (airwatch; imetrikus) in a diary twice daily for days after visit and days before visit . the visit procedures were repeated at day - and at baseline ( weeks or longer) after the onset of cold symptoms. definition of asthma exacerbation. before initiating the study, we defined asthma exacerbation as either a worsening of asthma symptoms that required corticosteroids (systemic or topical) or a worsening of symptoms on the basis of study measurements. the latter was defined by an increase in daily asthma symptom score of points or more (range, - points) above the baseline daily average for at least days together with at least of the following: ( ) decrease in fev by % or more at any of the first visits; ( ) decrease in peak flow measurements by % or more for at least days during the cold week; or ( ) increase in daily albuterol use of puffs or more for at least days during the cold week. specimen processing. nl specimens from visit (days - after the onset of cold symptoms) were analyzed for the presence of virus by pcr, viral culture, and virochip microarray analysis. for microarray analysis and pcr, ml of homogenized nl specimen was mixed with . ml of rlt buffer (qiagen) containing -mercaptoethanol and then frozen at bedside in dry ice and stored at Ϫ Њc. a . -ml nl aliquot was cultured in duplicate with a combination of different cell lines (hela, wi , mrc , primary monkey kidney, and hfdl, an in-house line of human fetal diploid lung cells [california department of health services viral and rickettsial disease laboratory, richmond]). some specimens were inoculated into all cell types, and all were cultured in at least , including primary monkey kidney and human fetal diploid. if cytopathic effect occurred, viral antigen detection tests (respiratory viral antigen detection kit; chemicon) were performed to identify antigens from adenovirus, rsv, influenza viruses a and b, and parainfluenza viruses , , and . enteroviruses and rhinoviruses were differentiated by reactivity with the monoclonal panenterovirus reagent from chemicon and the enterovirus monoclonal antibody from dako cytomation (enterovirus clone -d / ). rna extraction. samples were masked and total rna was extracted with an on-column recombinant dnase treatment step using the rneasy miniprep kit (qiagen), in accordance with the manufacturer's instructions. amplification and microarray analysis. rna was randomly amplified and labeled and was then hybridized to the virochip microarray as described elsewhere (protocol s in wang et al. [ ] ). microarrays were scanned using the axon b scanner and genepix software (version ; axon instruments). a.k., who was blinded to the viral culture isolation and hrv pcr results, interpreted the hybridization signatures. epredict, a computational tool developed for the virochip array hybridization signature, was used to determine the virochip results, with the p value cutoff for positivity set at . , as described elsewhere [ ] . samples for which multiple viruses met this threshold were further evaluated by hierarchical cluster analysis of sum-normalized background-subtracted array hybridization intensities from all nl specimens, by use of cluster software (version ) [ ] . co-occurrence of samples within clusters was used to make calls for specimens that either had multiple significant epredict scores or had no significant epredict scores. all viral-positive calls were confirmed by recovery of viral sequence. specific pcr for hrv detection. for each sample, ml of the randomly amplified material was used for independent pcr to detect and sequence the hrv vp /vp capsid gene junction, as described elsewhere [ ] . the vp /vp pcrs were performed in a blinded manner. primers -reverse ( -gcatciggyar-yttccaccaccancc- ; positions - of hrv- b; national center for biotechnology information [ncbi] accession number d ) and -forward ( -gggaccaactactt-tgggtgtccgtgt- ; positions - of hrv- b) were used for pcr ( cycles of Њc for s, Њc for s, and Њc for s). comparative sequence analysis of recovered hrv vp /vp pcr products. clustalw (version . ) was used to align the vp /vp capsid gene junction sequences obtained for the clinical isolates of hrv for all reference hrv serotypes [ ] . neighbor-joining phylogenetic trees were generated from the resulting alignment using the phylip package (version . ) [ ] . specific pcr for hcovs. for each of the hcov-positive samples identified by virochip microarray analysis, ml of the randomly amplified material was used for pcr to detect a bp region of the polymerase gene using pan-cov primers ( -ggttgggactatcctaagtgtga- and -ccatcatca-gatagaatcatcata- ) that have been described elsewhere [ ] ; amplification was with cycles of Њc for s, Њc for s, and Њc for s. sequence analysis of hcov pcr products amplified from clinical isolates. pcr products were extracted using qiaquick (qiagen) and were either sequenced directly using path-ogen-specific primers or subcloned into pcr . topo vector (invitrogen) and sequenced using m forward and m reverse primers with the bigdye cycle sequence kit on an abi automated sequencer (applied biosystems). the identity of each of the hcovs in the present study was inferred on the basis of the highest scoring match from blast analysis (version . . ) [ ] of the resulting sequences. amplified cdna was subcloned into pcr . topo plasmid (invitrogen). three hundred eighty-four colonies were picked, and dna was purified by magnetic bead isolation followed by dna sequencing using the bigdye cycle sequence kit/abi xl sequencer. sequence reads were assembled by use of consed for linux (version . ) [ ] . assemblies were screened by blast analysis [ ] to remove any contigs with human or bacterial sequence similarity. gaps in assemblies were filled by synthesis of oligonucleotides with at least bp of overlapping sequence with available contigs. accession numbers. genbank accession numbers for the sequenced viruses presented here are ef -ef . the geo database (http://www.ncbi.nlm.nih.gov/geo/) series accession number for all virochip microarray data presented here is gse . the first goal of our analysis was to assess the performance of the virochip relative to conventional viral detection methods. to do this, we used a set of nl specimens from an ongoing prospective study of naturally acquired upper rtis (naturis) in adults with and without asthma. a total of cold events captured from this study were available for anal- ysis. breakdown of participants, specimens, associated clinical outcomes, and corresponding virochip results are summarized in figure . each naturi specimen was analyzed independently in a blinded manner by distinct viral detection methods: virochip analysis and culture isolation for common respiratory pathogens (hrv; rsv; influenza viruses a and b; human parainfluenza viruses , , and ; adenovirus; and human enterovirus) and pcr for hrv. where the virochip detected viruses, follow-up pcr and sequence recovery was performed to confirm the presence of the detected viral species. a high proportion of specimens tested positive for virus by virochip analysis ( %). reflective of the outpatient setting of our study, the most prevalent virus families detected by the virochip corresponded to hrvs and hcovs (figure a and table ). four additional viruses generally thought to be associated with lower rtis-rsv and the closely related hmpv, influenza virus, and human parainfluenza virus-were also detected. we also detected double infections (rsv plus hcov and rsv plus influenza virus) ( figure a and table ). in contrast, virus was detected by culture isolation in only % of samples. three different viral pathogens (hrv, rsv, and influenza virus) and double infection were detected by this method (table ) . head-to-head comparison of these assays yielded an overall low concordance ( %). virtually all of the discordant results ( / ) corresponded to results that were positive by the virochip but negative by culture isolation. these results were not surprising, given the known limitations of viral culture isolation [ ] . we also compared the performance of the virochip to pathogen-specific pcr, which is known for its high sensitivity. to address this in a statistically significant manner, we focused on pcr detection of hrv [ ] , the most common pathogen detected in the study population. we found excellent concordance ( % agreement) between virochip and hrv-specific pcr results, indicating that virochip analysis is a highly sensitive ( %) and specific ( %) viral detection method in comparison to pathogen-specific pcr (table ). analysis of virochip hybridization signatures for the hcovs detected among the study subjects revealed distinct signatures ( figure b) . pcr recovery and sequence analysis of a fragment of the hcov polymerase gene [ ] revealed that each of these signatures corresponded to a distinct hcov subtype: ( ) hcov-oc , ( ) hcov-nl , and ( ) hcov-hku . each of these isolates shared between % and % sequence identity with the corresponding hcov polymerase gene sequences present in the ncbi database. surprisingly, the bulk of the hcovs detected in this outpatient population did not correspond to the more common hcov types (oc and e) historically detected in us adult rtis [ ] . of the hcovs detected in this study population, were hcov-nl , were hcov-hku , and only was hcov-oc . the different hcov types were detected in samples collected at different times during the study. all of the hcov-nl isolates were detected in persons with colds that occurred during the early winter of , whereas the hcov-hku isolates were detected during the late fall of ( isolate) and the early winter of ( isolates). the sole hcov-oc isolate was identified during the mid-fall of . no hcovs infections were detected in this study population in . interestingly, hcov-nl was detected only in persons with asthma, and of the infections were accompanied by an exacerbation of asthma symptoms. the most commonly detected virus in our study population was hrv (figures and a and table ). to investigate hrv diversity, we sequenced the junction of the vp /vp capsid genes of the hrv isolates and performed phylogenetic analysis of these sequences and of the published sequences from all hrv serotype reference strains [ ] . we found that the virochip hybridization signatures we observed corresponded to ∼ different hrvs: hrva serotypes, hrvb serotypes, and a novel third set of divergent hrvs (referred to as hrv'x'; figure ), which possessed slightly more sequence similarity to hrva than to hrvb reference serotypes. none of the divergent hrv'x' isolates were culturable; thus, unambiguous detection and classification of these isolates by conventional serotyping [ ] , drug susceptibility [ ] , or receptor-type usage assays [ ] was not possible. recovery of complete coding sequence from of the hrv'x' isolates (hrv'x'- and hrv'x'- ) and analysis of their sequence identity with a representative subset of fully sequenced hrva subgroup genomes and fully sequenced hrvb subgroup genomes [ ] indicated that these hrv'x' isolates were indeed hrvs. scanning pairwise identity revealed that the differences between the hrv'x' and the hrva subgroup genomes were not confined to a single locus but spanned the entire genome ( figure ) . although the vp /vp phylogenetic analysis indicated that the hrv'x' isolates were more similar to hrva than hrvb reference serotype strains, comparison of the level of genomewide sequence identity shared within the fully sequenced subset of hrva genomes to the levels of sequence identity shared between the hrva and the hrv'x' isolates showed that the hrv'x' isolates were almost as genetically distinct from hrva as the hrvb subgroup genomes ( figure ) . moreover, pairwise sequence identity between the hrv'x' genomes was much lower than that detected among the fully sequenced hrva or hrvb subgroup genomes (data not shown). taken together, these data demonstrate that these hrv'x' isolates correspond to a novel divergent subgroup of hrv and suggest that this divergent branch of hrv'x' isolates may possess a higher level of genetic diversity than seen previously in the hrva and hrvb subgroups. this is the first prospective study to use a pan-viral detection strategy to investigate the influence of viral pathogens on clinical outcome in rtis in persons with asthma. we find that, like pcr, the virochip technology is superior to standard culture isolation methods for detection of viral pathogens. moreover, the virochip exhibits comparable sensitivity and specificity to pathogen-specific pcr. on the whole, the distribution and proportion of distinct viral pathogens detected by the virochip agrees with previous pcr-based analyses of viral pathogens associated with upper rtis and those accompanied by exacerbation of asthma symptoms [ , ] . however, virochip analysis has allowed us to uncover a remarkable amount of diversity among the viral pathogens in this relatively small study population. this diversity indicates that future studies that seek to link a particular virus or set of viruses to a discrete clinical outcome, such as exacerbation of asthma symptoms, will need to include large numbers of subjects and use pan-viral detection methods (such as the virochip) that can differentiate among such isolates. two observations of viral diversity uncovered by virochip analysis of nl specimens derived from this study are particularly noteworthy. first, the diversity and distribution of hcovs detected in the present study were surprising. the more recently described hcov-hku and hcov-nl were the predominant hcovs rather than hcov-oc and hcov- e, which have been traditionally implicated in up to % of common colds in the us adult population [ ] . here, instead we see hcov-nl and hcov-hku making up approximately that same proportion of colds detected in our study population. given that hcov-nl and hcov-hku have not been implicated previously as significant players in outpatient respiratory tract illnesses among immunocompetent adults in the united states [ , ] , these results were unexpected. no hcov- e isolates and only a single hcov-oc isolate were detected in this study group, despite the fact that independent studies with the virochip have demonstrated that, when present, both hcov-oc and hcov- e are readily detectable (c. y. chiu, a. urisman, t. l. greenhow, submitted). further analysis will be required to determine whether the patterns of hcovs detected here reflect an increased susceptibility of adults with asthma to contract these hcovs, the arrival of a local outbreak of these hcov types, or an actual shift in the prevalence of the distinct hcovs circulating in the us adult population. second, the virochip detected remarkable and unanticipated diversity among hrv isolates. in addition to detecting almost distinct hrv species closely related to known reference hrv serotypes, we also identified a subgroup of genetically distinct hrvs in a significant fraction ( / ) of the clinical isolates of hrv. although the clinical significance of hrv diversity remains incompletely understood, the detection of such a high level of genetic divergence among hrv strains captured in this relatively small study population indicates that the standard hrv reference serotypes (which were characterized almost years ago) do not adequately describe the diversity of currently circulating hrvs. we do not believe that these hrv'x' strains are an anomalous subgroup of hrvs unique to this study population because ( ) a similar proportion of divergent hrv strains were detected by virochip analysis in an unrelated cohort of pediatric subjects with rtis (c. y. chiu, a. urisman, t. l. greenhow, submitted); ( ) divergent hrvs have also been recently identified in a study of pediatric respiratory infections in australia [ ] ; ( ) based on the noncoding region sequence alone, virtually identical isolates of hrv have been reported in independent analyses of hrv infections among europeans [ ] ; and ( ) recent independent application of a distinct pan-viral detection tool, masstag pcr analysis, has also documented a set of highly diverged hrvs circulating in the us population [ ] . comparison of the vp sequences of the hrv'x' strains identified here suggests that one of the hrv'x' strains (hrv'x'- ) possesses high sequence similarity ( % identity with vp sequence dq ) to one of the divergent hrvs recently reported by lamson et al. [ ] . however, the vp sequences in the other hrv'x' strains identified here possess ! % nucleotide sequence identity with the set of divergent hrvs identified by lamson et al. [ ] . the identification of distinct sets of genetically divergent hrv clinical isolates indicates that the set of previously unrecognized hrvs currently circulating in the united states may be quite large. a deeper knowledge of the extent of the current hrv diversity should inform future studies of the role played by hrv strains in asthma exacerbations, given that a high proportion of such events are attributable to infection by these agents. in sum, the present data demonstrate that the virochip captures the entire spectrum of known respiratory viral pathogens in a single test, exhibits excellent sensitivity, and provides the capacity to identify as-yet-undiscovered agents. the application of the virochip to prospective clinical studies should enable us to develop a comprehensive picture of the diversity of viral pathogens present during infection, a critical missing piece of the puzzle required to advance our understanding of how different viral pathogens influence the course and spectrum of disease. the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis community study of role of viral infections in exacerbations of asthma in - year old children viruses in asthma respiratory viruses and exacerbations of asthma in adults the incidence of respiratory tract infection in adults requiring hospitalization for asthma viruses in asthma exacerbations prechallenge antibodies: moderators of infection rate, signs, and symptoms in adults experimentally challenged with rhinovirus type rhinovirus- colds in healthy and in asthmatic subjects: similar changes in upper and lower airways relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection the role of t lymphocytes in asthma rhinovirus inhalation causes long-lasting excessive airway narrowing in response to methacholine in asthmatic subjects in vivo holgate pan-viral screening of adult rtis • jid lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects effects of experimental rhinovirus infection on airway hyperresponsiveness to bradykinin in asthmatic subjects in vivo experimental rhinovirus infection: effects on cell differentials and soluble markers in sputum in asthmatic subjects effect of experimental rhinovirus colds on airway hyperresponsiveness to histamine and interleukin- in nasal lavage in asthmatic subjects in vivo exacerbations of asthma in adults during experimental rhinovirus infection peak expiratory flow changes during experimental rhinovirus infection experimental rhinovirus infection causes variable airway obstruction in subjects with atopic asthma microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays microarray detection of human parainfluenzavirus infection associated with respiratory failure in an immunocompetent adult identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r q rnasel variant diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a panvirus microarray the wisconsin upper respiratory symptom survey is responsive, reliable, and valid e-predict: a computational strategy for species identification based on observed dna microarray hybridization patterns cluster analysis and display of genome-wide expression patterns genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus phylip-phylogeny inference package (version . ) characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia basic local alignment search tool consed: a graphical tool for sequence finishing the common cold coronavirus infections in working adults: eight-year study with e and oc antigenic groupings of rhinovirus serotypes two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity many rhinovirus serotypes share the same cellular receptor genome-wide diversity and selective pressure in the human rhinovirus rhinovirus and the lower respiratory tract coronavirus hku infection in the united states human coronavirus nl , a new respiratory virus characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during we are grateful to shoshannah beck for providing technical support with sample processing and microarray analysis for a subset of the specimens and to anatoly urisman, kael fischer, charles chiu, and patrick tang for assistance, advice, and technical input throughout the course of this study. key: cord- -hap flng authors: arruda, eurico; cintra, otavio a.l.; hayden, frederick g. title: respiratory tract viral infections date: - - journal: tropical infectious diseases doi: . /b - - - - . - sha: doc_id: cord_uid: hap flng nan acute respiratory infections (aris) are prevalent worldwide and rival diarrhea as the leading cause of death in developing countries. , in some impoverished urban populations in south america, ari symptoms may be present on an almost continuous basis, making it difficult to determine symptom-free days and estimate attack rates. , children from these areas may spend % to % of their time with respiratory symptoms, , mostly caused by upper respiratory infections (uris). the most striking disparity between developing and developed countries with regard to ari epidemiology is the case-fatality rate of lower respiratory infection (lri), mainly pneumonia, bronchiolitis, and influenza, , in children under years of age, which may reach % in some areas. , several community-based studies have established the importance of common respiratory viral infections in tropical countries (table - ) . in impoverished populations, these common viral infections may occur simultaneously with measles, diarrhea, and malnutrition, resulting in complex interactions of pathologic conditions that carry the potential to become life-threatening diseases. , unlike certain pathogens restricted to tropical areas, the respiratory viruses have worldwide distribution, efficient person-to-person transmission, and an impact on all age groups. except for a few agents (e.g., adenoviruses, severe acute respiratory syndrome [sars] coronavirus) and rare cases of extrapulmonary dissemination with other respiratory viruses, replication is generally restricted to the respiratory mucosa of humans. in most health-care facility-based studies of acute lri (alri) conducted in tropical countries (table - ), respiratory syncytial virus (rsv) is the virus most frequently detected ( % to %), followed by parainfluenza viruses ( % to %), adenoviruses ( % to %), and influenza viruses ( % to %). with few exceptions, human rhinovirus (hrv) and human coronaviruses (hcov) have not been reported frequently in studies in tropical countries, probably because of difficulties in their detection. although previous studies have shown that attending daycare centers can be a risk for ari, providing day care for children has become an important economic issue in developing countries, where mothers must join the workforce to contribute to the family income. consistent with studies in the united states and elsewhere, a study found a high burden of ari in young, low-income children attending day care in salvador, brazil. few specific interventions are available to reduce the impact of respiratory viruses, and the application of these interventions may be further hampered by epidemiologic patterns in ari and socioeconomic differences in temperate, developed countries compared with equatorial regions. for example, housing conditions and crowding pose challenges for optimizing health-care strategies in the tropics. while the incidences of some respiratory viruses, particularly rsv and influenza, show seasonal trends in some tropical areas, the association of seasonal peaks of respiratory viruses in general may be less apparent where fluctuations in temperature are smaller. nutritional and educational interventions, such as reinforcing breast-feeding, vitamin a supplementation for measles, and facilitation of access to oral rehydration therapy, may have significant effect on the morbidity and mortality due to lri alone or in association with diarrhea. in this chapter we focus attention on the most common viral respiratory infections, whose main features are summarized in table - , and try to highlight features unique to the developing world. in tropical countries influenza activity may occur yearround as well as in outbreaks more typical of temperate regions. these infections cause serious disease in populations weakened by malnutrition, with limited access to medical care. of note, the predisposition induced by influenza to superimposed bacterial infections, mainly streptococcus pneumoniae, may greatly affect morbidity and mortality, mainly among impoverished populations. in addition, influenza viruses can reassort or sometimes cross species barriers to generate emergent strains that may cause localized outbreaks or potentially pandemics with enormous impact for health on a global scale. influenza viruses are pleomorphic, enveloped, with segmented negative-strand rna genomes and belong to the family orthomyxoviridae. influenza viruses are distributed in three genera-a, b, and c-based on the antigenicity of the nucleoprotein (np) and matrix protein. influenza a virus is further classified in subtypes based on its two surface glycoproteins: hemagglutinin (ha) and neuraminidase (na). among the ha and na subtypes recognized in nature, ha (h , h , h , h , h , and h ) and three na (n , n , and n ) subtypes have now been identified in human isolates of influenza a viruses. however, only three subtypes of ha (h , h , and h ) and two of na (n and n ) have caused pandemics and sustained circulation in human populations in recent years. the genomes of influenza viruses contain eight rna segments in influenza a and b viruses, and seven rna segments in influenza c. the glycoprotein ha is responsible for the attachment of the virus to sialic acid-containing cell receptors, and it mediates fusion and penetration. proteolytic cleavage of ha by cellular serine proteases exposes hydrophobic fusion domains that mediate membrane fusion. the na cleaves terminal sialic acid from glycoconjugates present on respiratory mucins, cells, and progeny virions. this action destroys receptors recognized by ha and allows budding virus to be released from infected cells and to spread within the respiratory tract. influenza c virus contains a single surface glycoprotein that binds to receptor, promotes fusion of membranes, and also cleaves sialic acid. virus binding to receptor is followed by internalization into endosomes, fusion of viral and endosomal membranes, and release of the genome to the cytoplasm, from where it is transported to the nucleus. in influenza a viruses, the m protein serves an ion channel function that facilitates dissociation of the rna segments from the virion interior. transcription of the negative-strand genomic rna into positive-strand messenger rna (mrna) and complementary rna (crna) is mediated by a viral rna polymerase complex in the nucleus. crna serves as a template for the synthesis of negative-strand virion rna genome segments, and mrna directs viral protein synthesis. newly assembled nucleocapsids acquire an envelope as they bud through the cell surface. only viruses with a full complement of genome segments are infectious. influenza a viruses are primarily viruses of aquatic birds, particularly ducks and shore birds, which harbor all subtypes recognized to date. selected subtypes naturally infect a range of terrestrial (swine, horses, humans) and aquatic (seals) mammals; influenza b virus infects humans and uncommonly seals, dogs, cats, and swine, and influenza c virus is primarily a virus of humans. depending on the virus type and subtype, experimental infection can be induced in mice, ferrets, chickens, swine, and primates, and the viruses can be propagated in primary cultures of kidney cells, continuous cell lines (mdck, vero, per.c , and llc-mk ), and also in embryonated hen' s eggs. the biologic property of influenza viruses to bind erythrocytes is exploited for early detection of the virus in cell culture and for the development of serologic assays by hemagglutination inhibition. influenza viruses are inactivated by temperatures above °c and by lipid solvents, acid, formaldehyde, ionizing radiation, and ultraviolet (uv) light. influenza viruses occur throughout the world, causing highly contagious respiratory infections with high morbidity and excess mortality (in seasonal peaks), particularly in infants and the elderly. in developing tropical countries, influenza has been associated with an average of % of aris leading to physician contact. , this apparently low proportion probably represents only the most severe cases, since % to % of children under years of age in tropical africa have been found to seroconvert in one outbreak. previously healthy children younger than year of age are hospitalized for influenza at rates similar to those for adults at high risk for influenza, and influenza accounts for a great number of outpatient visits and courses of antibiotics in children of all ages. when human influenza virus is introduced into a malnourished population with limited access to care, high morbidity and mortality rates can occur, as was observed in madagascar in where a conventional influenza a/h n subtype virus was associated with case-fatality rates of approximately %. contrary to the remarkably sharp seasonality of influenza a outbreaks in temperate countries, seasonal patterns in tropical countries have varied between studies. in southern india and thailand influenza has occurred throughout the year with sporadic outbreaks, whereas there have been consistent outbreaks in june-july and november-january, coinciding with the winter seasons in the southern and northern hemispheres, but with no apparent association with meteorologic factors. in the philippines influenza has been more frequent between november and january, while in senegal, nigeria, and taiwan there has been clear association with increased rainfall. in southeastern brazil, argentina, as well as in south africa, seasonal outbreaks of influenza a have occurred annually from may through august (mid autumn through winter) in association with cooler temperatures but not with rainfall. influenza b outbreaks occur periodically, yet less frequently than influenza a, in both temperate and tropical regions, , whereas influenza c is generally nonseasonal. minor changes in antigenicity, called antigenic drift, are caused by accumulation of point mutations in the genes coding for influenza ha and na, generating new strains that spread in annual epidemics. influenza viruses b and c are less prone to antigenic drift. major antigenic changes in influenza a are called antigenic shift and result in the emergence of a novel ha subtype with or without new na to which humans lack significant immunity. this may be caused by the acquisition of new gene segments through genetic reassortment in a host infected simultaneously by a human and an animal (typically avian) virus, or by reappearance of a subtype from reservoir. swine are susceptible to both avian and human influenza viruses and may be hosts for reassortment or serve as the mammalian species in which avian viruses can adapt. novel influenza virus subtypes generated by shift have caused catastrophic pandemics, including three in the last century. the h n "spanish flu" pandemic of is estimated to have caused up to million deaths worldwide, while the h n "asian flu" in and the "hong kong flu" in caused an estimated to million deaths. recent clusters of human infections due to avian influenza, particularly h n subtype viruses in asia, have raised concerns about new pandemic threats. in a highly pathogenic avian influenza h n virus resulting from reassortment among several avian viruses caused lethal outbreaks in domestic poultry and severe illness with deaths among human cases in hong kong. the outbreak was due to exposure to infected poultry in live poultry markets and was later contained by their slaughter. this virus was transmitted inefficiently from person to person. , in february an h n virus caused deaths in a family visiting fujian province in china. since late wide-scale poultry outbreaks due to h n virus have occurred in at least asian countries. interspecies transmission to humans occurred in at least two countries, causing over cases and deaths in vietnam and thailand. these h n viruses have continued to reassort, evolve antigenically, and extend their host range with documented infections in swine and felids. prolonged nonsymptomatic excretion in ducks and detection in migratory birds indicate that this virus has become endemic in southeast asia. , an avian h n virus with human receptor specificity has also spread throughout asia in domestic poultry and pigs and caused mild disease in humans in hong kong and china. an avian h n virus caused conjunctivitis in at least people and one fatal pneumonia in the netherlands in ; the outbreak was contained by culling and quarantine of poultry. in february a highly pathogenic h n virus emerged in domestic poultry in british columbia with two documented human cases of conjunctivitis and mild "flulike" illness. , influenza virus is transmitted from person to person by large droplets and small-particle aerosols, as well as possibly by fomites with hand contamination and subsequent selfinoculation. the relative importance of these routes is uncertain for natural influenza. ingestion of infected birds has led to infection by avian viruses in cats. secondary attack rates may reach over % in semiclosed populations, especially among schoolchildren and patients debilitated by underlying conditions who live in relative confinement, such as nursing home residents. children play a major role in influenza outbreaks with respect to propagation of the epidemic virus in families and communities. classic influenza starts abruptly after an incubation period of to days, with fever, chills, malaise, headache, myalgia, and prostration, often accompanied by nonproductive cough, sore throat, and mild rhinorrhea. systemic complaints last to days, whereas sore throat, hoarseness, and cough, with substernal discomfort, may increase in severity as the systemic symptoms subside. cough and asthenia often persist for weeks or longer. respiratory symptoms may be minimal or absent initially, especially in the elderly or infants. in frail elderly persons, lassitude, lethargy, confusion, lowgrade fever, and sometimes gastrointestinal complaints may be the primary findings. influenza b tends to be milder than influenza a, and influenza c typically causes colds or bronchitis. influenza may also present as unexplained fever, croup (laryngotracheobronchitis), vomiting, diarrhea, and neurologic manifestations in young children. up to % of influenza virus infections in adults are subclinical. influenza causes a variety of viral respiratory complications, including otitis media, sinusitis, tracheobronchitis, pneumonia, and, in young children, bronchiolitis and croup. secondary bacterial infections, especially pneumonia caused by staphylococcus aureus, streptococcus pneumoniae, and haemophilus influenzae, are common complications and should be suspected in relapses of fever, chest pain, and cough. influenza is also associated with invasive meningococcal infections. other complications include exacerbations of asthma, chronic bronchitis, and congestive heart failure. myositis, myoglobinuric renal failure, meningoencephalitis, transverse myelitis, polyneuritis, parotitis, myocarditis, arthritis, and disseminated intravascular coagulation rarely occur after influenza. reye' s syndrome occurs in fewer than per , cases of influenza in patients under years of age following the use of salicylates. pregnant women, human immunodeficiency virus (hiv)-infected patients, and other immunocompromised hosts are at higher risk for severe disease and complications. the virus infects the respiratory mucosa, where it causes lytic infection of cells and desquamation of the respiratory epithelium, mononuclear cell infiltrates in the lamina propria, and altered mucociliary clearance. tracheobronchitis is a typical feature and often associated with prolonged abnormalities in small airway pulmonary function and airway hyperreactivity. primary influenza viral pneumonia results in diffuse alveolar damage, alveolar hemorrhage and exudate, hyaline membranes, and later reactive fibrosis. fatal cases show pathologic changes in nonrespiratory organs, such as brain congestion and swelling, myocardial inflammation, and fibrinoid changes in arterioles. viral replication in the upper respiratory tract generally peaks within or days of symptom onset and, depending on age and prior immunologic experience, continues for about to days. the severity of illness broadly correlates with upper respiratory tract viral levels. constitutional symptoms with influenza are due in part to the release of proinflammatory cytokines and chemokines. levels of interferon (ifn-α and ifn-γ), tumor necrosis factor (tnf-α), interleukins and chemokines (il- β, il- , il- , il- , mcp- , mip- α and mip- β) are increased in nasal secretions, and ifn, il- , and tnf-α are increased in blood in human influenza. the tissue tropism of a strain of influenza virus depends, among other factors, on a combination of susceptibility of its ha to be cleaved by, and tissue availability of proteases with specificity to cleave it, thus rendering the virus infectious. extrapulmonary dissemination of virus has been uncommonly documented in humans, but systemic spread is a regular feature of highly pathogenic avian viruses in chickens and sometimes in rodents or other mammalian hosts. serum and secretory antibodies directed to ha and na appear about days after infection. protection against reinfection by the homologous strain is durable following natural infection and is correlated with serum and nasal neutralizing antibody levels, principally directed against ha. vaccine-induced protection may last for up to to years against homotypic virus. infection also induces cell-mediated immunity, which is detectable to days after infection and seems to be important for recovery. cytotoxic t-lymphocyte responses against internal proteins may provide some degree of heterosubtypic immunity. the diagnosis of influenza is frequently made on the basis of clinical and epidemiologic information. a higher index of suspicion and laboratory diagnostics is needed outside the season, particularly in sporadic individual cases or unexplained outbreaks of febrile respiratory illness. viral isolation from respiratory specimens can be done in several types of cells (e.g., prmk, mdck, llc-mk ) and remains the current standard. the presence of virus may be detected in cell cultures by hemadsorption with guinea pig erythrocytes before or after cytopathic effect (cpe) is visible. blind hemadsorption is positive days after inoculation in almost all positive samples. confirmation of isolates can be done by hemagglutination inhibition or immunofluorescence with typespecific antisera. diagnosis can also be made in to days by immunofluorescence of monolayers of mdck cells inoculated by centrifugation (shell-vial). conserved influenza antigens (m or np) directly in clinical samples can be detected by one of several techniques (e.g., immunofluorescence [if], enzyme immunoassay [eia]), and multiple point-of-care kits are commercially available with turnaround time of to minutes. one commercial assay is based on detection of influenzaspecific na activity. the sensitivities of these assays are higher in children (up to %) than in adults (generally % to %) and depend on duration of illness and sample type. several formats of reverse transcription-polymerase chain reaction (rt-pcr) assays have been used for the detection of influenza a and b rnas in clinical samples, with the advantage of detecting genomes of noninfectious virus. the time to perform rt-pcr is longer but the cost may be lower than for commercial rapid antigen detection kits, especially in developing countries. real-time rt-pcr has enabled the development of assays that provide rapid quantitative detection of influenza a and b with high sensitivity. , these assays have great potential to replace other methods, because they are simultaneously rapid, highly sensitive, quantitative, and amenable to being used in multiplex format, which might include probes for several different respiratory pathogens. however, the costs are still prohibitive for most laboratories in developing nations. serologic diagnosis of influenza using paired acute and convalescent serum can be done retrospectively by a variety of techniques but mainly for serologic survey purposes. amantadine and rimantadine are m ion channel blockers that inhibit influenza a virus replication at the uncoating step. in uncomplicated influenza a in adults without underlying diseases, treatment with either drug can reduce the duration of influenza illness by approximately to days if started early, within hours from the onset of symptoms. amantadine is excreted in an unchanged state in the urine, while rimantadine is extensively metabolized after absorption and less than % of the dose is excreted unchanged in the urine. elderly persons need only half the dose to achieve similar plasma levels. amantadine or rimantadine may cause gastrointestinal upset and central nervous system side effects. central nervous system (cns) intolerance is more common with amantadine and, when severe, can be manifested as agitation, psychosis, seizures, and coma. mild complaints including insomnia, dizziness, anxiety, dry mouth, anorexia, and nausea are reversible upon discontinuation. amantadine and rimantadine are marketed as -mg tablets and -mg/ml syrup. the recommended dose is mg twice daily for adults older than years of age ( mg/day for patients ≥ years of age). for children under age years, a rimantadine dose of mg/kg/day (maximum, mg/day) has been suggested. dose reductions proportional to the creatinine clearance (clcr) are suggested for patients with renal insufficiency (amantadine for clcr < to ml/min/ . m ; rimantadine for clcr < to ml/min/ . m ). influenza virus resistant to amantadine-rimantadine emerges in approximately one third of treated patients; such viruses are transmissible to close contacts and cause typical influenza illness. resistance to these drugs renders them ineffective and is sometimes present naturally, including in recent human isolates of h n virus. the neuraminidase inhibitors zanamivir and oseltamivir inhibit both influenza a and b viruses by blocking the active site of the enzyme for cleavage of sialic acid, thus inhibiting virus release from infected cells and spread within the respiratory tract. in adults and children older than years, inhaled zanamivir ( mg twice daily for days) provides to . -day reduction in illness and reduces antibiotic use for lower respiratory complications by %. zanamivir is generally well tolerated but may uncommonly induce bronchospasm, particularly in those with influenza and pre-existing airway disease. oseltamivir ( mg orally twice daily for days) reduces illness severity, time to resumption of daily activities by to days, and rates of complications leading to antibiotic prescription and hospitalization by about % in adults. in children to years of age, oseltamivir reduces the frequency of otitis media and, consequently, antibiotic prescriptions. side effects include mild-to-moderate nausea or emesis. dosage of neuraminidase inhibitors does not need to be adjusted for the elderly. resistance emergence is uncommon with both drugs, although a recent study of children treated with oseltamivir detected drug-resistant viruses in %, often in association with prolonged viral excretion, and showed that children can be a source of viral transmission, even after days of treatment. antipyretic-analgesic drugs may be used for influenzainduced fever and aches. aspirin should be avoided because of its association with reye' s syndrome. immunization with formalin-inactivated or live-attenuated multivalent influenza virus vaccines and chemoprophylaxis for influenza virus a are the methods available for preventing influenza. influenza vaccine is used prior to the influenza season and currently includes one strain of influenza b and two strains of subtypes h n and h n of influenza a virus, chosen by the world health organization (who) surveillance network among the viruses most likely to circulate in the next influenza season. , the inactivated vaccine has an approximate % to % efficacy in preventing illness in healthy children and adults. it also reduces influenza-related hospitalizations and mortality in elderly and high-risk patients. the centers for disease control and prevention (cdc) recommends the immunization of persons aged years and older; residents of nursing homes; children and adults with chronic cardiovascular or pulmonary disease, including asthma; persons chronically ill with diabetes mellitus, renal dysfunction, or hemoglobinopathies; immunosuppressed patients including those with hiv infection; children and adolescents on chronic aspirin therapy who may develop postinfluenza reye' s syndrome; women who will be pregnant during the influenza season; children aged to months; those who can transmit influenza to persons at high risk, such as health-care workers and household contacts of those at high risk including children to months of age; crew members of cruise ships; providers of essential services; and unimmunized travelers to areas where influenza may be circulating, including the tropics, the southern hemisphere between april and september, and those traveling in large organized tourist groups. in addition, vaccine is made available to anyone interested in reducing the likelihood of becoming ill with influenza. , the inactivated vaccine, administered as a single intramuscular (im) dose shortly before influenza season (two doses in previously unimmunized children < years of age), is safe during pregnancy but should be avoided in persons with history of anaphylactic reactions to eggs. vaccine safety and efficacy in children has been extensively evaluated and has shown a favorable safety profile with efficacy in -to -year-old children of % to %. inactivated vaccine is not currently recommended for children younger than months, but vaccination of household contacts and caregivers should reduce the risk of these high-risk children contracting influenza. healthy people aged to years who are not contacts of immunosuppressed patients can receive either inactivated or intranasal live-attenuated vaccines. influenza inactivated vaccine has been recently introduced in many tropical areas of the world, with a composition based on influenza viruses circulating in the southern hemisphere. the vaccine is given prior to the influenza season, which for most countries in the southern hemisphere is between may and july. in south america, annual vaccination of the elderly has reduced hospitalizations and mortality for respiratory diseases. continuous surveillance has already shown that regional variations of circulating influenza virus strains should be taken in consideration in the formulation of influenza vaccines with compositions more appropriate for south america. live-attenuated, cold-adapted vaccines administered intranasally are well tolerated, genetically stable, and rarely transmissible and have the advantage of inducing local secretory immunoglobulin a (iga) responses. because of potential interference between components, two doses may be required in young children. this vaccine was licensed in the united states in , where it has become an option for healthy persons aged to years, including those in close contact with groups at high risk and those wanting to avoid influenza. this vaccine is not recommended for persons with asthma and other chronic disorders of the pulmonary or cardiovascular systems; persons with underlying medical conditions, including diabetes, renal dysfunction, and hemoglobinopathies; persons with known or suspected immunodeficiency diseases or who are receiving immunosuppressive therapies; children or adolescents receiving aspirin or other salicylates; persons with a history of guillain-barré syndrome; pregnant women; and persons with a history of hypersensitivity to eggs. cold-adapted trivalent influenza vaccine is highly effective ( % in phase studies) in preventing cultureconfirmed influenza in healthy children and has provided protection against drift variant strains in some studies. in young and middle-aged adults, efficacy is generally comparable to that of inactivated vaccine. other investigational approaches have been explored in influenza vaccine development, including recombinant ha produced in insect cells, virosomes incorporating surface glycoproteins, m protein conjugated with hepatitis b virus core, and naked dna encoding influenza virus nucleoprotein or ha. cell culture-based vaccines (mdck, vero) have been approved in europe and may offer an alternative to the limitations of the current egg-grown vaccines. the technique of reverse genetics has been used to rapidly produce candidate vaccines against potential pandemic threat viruses. amantadine and rimantadine are approved for use, and are % to % effective in the prophylaxis of influenza a during outbreaks. unvaccinated elderly persons, immunodeficient patients, patients in chronic care institutions experiencing outbreaks, persons who could not be vaccinated, and those who received a vaccine strain different from the outbreak strain may receive prophylaxis with amantadine or rimantadine. prophylaxis should be started as early as possible at doses equivalent to those used for therapy, and continued until week after the end of the outbreak for a total of at least weeks. amantadine-and rimantadine-resistant mutants of influenza a virus occur in up to % of treated patients and may be associated with failure of drug prophylaxis. both oseltamivir ( mg twice daily) and inhaled zanamivir ( mg/dose twice daily) are more than % effective in the prophylaxis of influenza during outbreaks, but only oseltamivir has been approved for this indication in the united states. , antiviral agents, especially the neuraminidase inhibitors, could significantly help in the control of a future influenza pandemic by reducing lower respiratory complications and hospitalizations as well as potentially person-to-person transmission. however, supply limitations pose a real difficulty. therefore, policies to ensure a reasonable supply of these drugs, as well as directions to optimize the use of limited supplies, are important issues to be considered. respiratory syncytial virus (rsv) is the single most important viral cause of lower respiratory disease and a major cause of morbidity and mortality in children worldwide. rsv is the leading cause of hospitalization in young children in developed and developing countries. in tropical areas, rsv has been the most frequently isolated virus in hospital-based ari studies of children. agent rsv, the only known human pathogen of the genus pneumovirus in the family paramyxoviridae, is a pleomorphic rna virus with helical nucleocapsid and lipid-containing envelope. antigenic differences in the surface glycoprotein g permit the classification of rsv into groups a and b, each with antigenic subgroups. the interaction of rsv envelope glycoprotein g with glycosaminoglycans enables adherence to the cell surface. however, g protein-independent mechanisms of attachment must exist, since mutants devoid of g protein can also enter host cells. rsv enters the cell by fusion of viral envelope with cell membranes, a process mediated by binding of the viral f protein to the cell gtpase rhoa. the syncytia resulting from fusion of the infected cells to adjacent ones are the major feature of the cytopathic effect of paramyxoviruses. once in the cytoplasm, the negative-strand rna is transcribed by viral transcriptase into mrnas, which then direct viral protein synthesis. an intermediate positivestrand full-length crna serves as a template for the synthesis of progeny negative-strand rna. as they bud through the cell membrane, the virions acquire a glycoprotein-containing envelope. rsv causes asymptomatic infection in a variety of experimental animals, but natural infection occurs only in humans and chimpanzees. rsv grows well in several human heteroploid cell lines, such as hep- , hela, and a , and is sensitive to ether, chloroform, detergents, and a ph less than . rsv is inactivated at °c, survives poorly on porous surfaces, and loses infectivity significantly by slow freezing and storage at temperatures above °c. rsv occurs worldwide and causes annual outbreaks in temperate climates in the winter and early spring, with sporadic cases throughout the year. in tropical regions, where temperature fluctuations are smaller and the only significant seasonal variable is often rainfall, rsv outbreaks tend to occur in the rainy seasons. such has been the case in malaysia, hong kong, india, papua new guinea, colombia, kenya, and the gambia. interestingly, in singapore rsv peak activity occurs from march to august, a period of higher temperature, higher day-to-day temperature variation, and lower relative humidity. in southeast brazil, rsv occurs seasonally, within a broader range of months from february through july, after the rainy season and when temperatures tend to be cooler, with slight variations from year to year. in regions where average winter temperatures are colder, such as in são paulo city and the southernmost parts of brazil, as well as in argentina, rsv peak activity tends to occur in july and august. [ ] [ ] [ ] most children have specific serum rsv antibody by age years, but reinfections occur throughout life. more than one subtype of either rsv group may cocirculate in one season, with group predominance changing from year to year, without apparent correlation with clinical or epidemiologic characteristics of the illness they cause. , rsv transmission requires close contact and occurs either by large-particle aerosols or by contamination of hands and inoculation into the eye or nose. secondary infections in family contacts of an index case are common, after an average incubation period ranging from to days. it is estimated that % of all infants will have rsv infection that requires medical attention and that % of them will be hospitalized. an estimated % of children will have bronchiolitis in their first year of life, with % to % of those infections caused by rsv. in southeast brazil, rsv is the leading cause of lower respiratory tract infections in children younger than year of age and is responsible for up to % of hospitalizations in this age group during peak months. the spectrum of illnesses caused by rsv ranges from mild uri to severe lri, including pneumonia, bronchiolitis, tracheobronchitis, and croup. in infants and young children, uri with fever and otitis media is common. during outbreaks, rsv rna has been detected in up to % of middle ear effusions in children with rsv infection and acute otitis media. the most frequent lri caused by rsv in infants is bronchiolitis, usually preceded by to days of uri symptoms, and progressing to lower respiratory tract involvement characterized by tachypnea, dyspnea, cough, expiratory wheezing, air trapping, and in more severe cases, intercostal muscle retractions and cyanosis. fever is present in only % of infants. chest radiographs may show hyperaeration of the lungs and sometimes segmented atelectasis. blood counts usually show lymphocytosis, and an increase in neutrophils with a left shift could be associated with bacterial superinfection. the most frequent bacterial superinfection in children with rsv infections is acute otitis media, which may be found in up to % of children with brochiolitis. however, more serious bacterial infections that may require sepsis work-up is uncommon in previously healthy infants with rsv infections. this may be different, however, in developing tropical areas, where rsv frequently causes infections in children previously debilitated by other diseases and malnutrition. infants with congenital heart disease, premature infants, or infants with underlying pulmonary conditions, such as cystic fibrosis and bronchopulmonary dysplasia, as well as immunocompromised hosts of any age, are at risk for severe and fatal rsv infections. hiv-infected children with rsv infections have a higher rate of pneumonia and prolonged illness and virus shedding, but the general severity of the rsv disease is not increased. differential diagnosis of acute bronchiolitis includes asthma, pneumonia, congenital heart and lung diseases, and cystic fibrosis. particular clinical signs are generally not accurate predictors of specific viral causes, but in a study conducted in the philippines, wheezing was a significant predictor of viral lri, while manifestations of higher severity, such as chest indrawing and cyanosis, were more often associated with bacterial lri. the most frequent rsv illness in children over years of age and adults is uri with coryza and cough, sore throat, and hoarseness, often accompanied by low-grade fever. exacerbations of chronic pulmonary diseases and wheezing can also be seen in adults with rsv infection. the role of rsv infections in causing wheezing and asthma exacerbations in infants is well established in studies conducted in temperate areas. similar observations have been made in an emergency room study conducted in southeast brazil, which found that infection with respiratory viruses, especially rsv, and a family history of allergy were independently associated with wheezing. similar findings have been observed in urban nigerian preschool children. rsv has been increasingly recognized as a cause of lri in the elderly, mainly characterized by interstitial pneumonia, prolonged cough, and dyspnea in persons with chronic pulmonary conditions, and it should be considered in the differential diagnosis of flulike illnesses. rsv replicates in respiratory epithelium to reach titers as high as tcid /ml in nasal secretions of infected babies, and virus shedding may be as prolonged as weeks after the symptoms disappear. rsv spreads from cell to cell and may involve the entire respiratory tree, reaching bronchioles in to days after the onset of rhinorrhea. replication in the bronchiolar epithelium causes necrosis of ciliated cells, syncytia formation, peribronchiolar inflammation with abundant lymphocytes and macrophages, and impairment of secretion clearance, resulting in small airway obstruction and the hyperinflation characteristic of bronchiolitis. pneumonia frequently coexists, evidenced by interstitial mononuclear infiltrate, eosinophilic cytoplasmic inclusions in epithelial cells, and multinucleated giant cells. the most severe rsv disease occurs in young babies, whose immature airways may be unable to compensate for the pathologic changes. naturally acquired immunity to rsv is incomplete and short-lived, but the severity of illness tends to decrease with reinfections. local secretory iga correlates better with protection than does serum antibody level and age, and pre-existing virus-specific maternal antibodies influence the development of neutralizing antibodies. cell-mediated immune response is central to recovery from rsv infection, and patients with suppressed cell-mediated immune response are at risk of severe rsv pulmonary disease and fatal outcome. , the type of immune response to the virus is probably a major factor in the development of wheezing and asthma exacerbations. a bias toward a th cytokine response seems to be associated with more severe disease, whereas a th response leads to effective viral clearance and milder illness. the virus itself generally triggers a th response, but a preexisting th deficiency may be associated with disease severity in some children. it has been suggested that rsv bronchiolitis may be a marker of predisposition to wheezing or asthma later in life. , children vaccinated with a formalin-inactivated rsv vaccine developed in the s had severe disease when exposed to natural infection, apparently as a consequence of an imbalance between protective and immunopathologic t-cell responses elicited by previous parenteral immunization with inactivated rsv. this would favor a cd + th cytokine pattern in response to subsequent rsv infections, whereas a previous natural infection would favor a cd + th pattern in response to reinfection. nasopharyngeal aspirates or swabs, nasal washings, and lower respiratory samples are all appropriate specimens for rsv isolation. this is usually accomplished in cultures of hep- cell line, in which rsv induces syncytia in to days. rsv antigen detection by eia, including membrane-based eia, is sensitive and specific and requires virtually no equipment, making it ideal for field studies. rapid rsv detection by if of exfoliated respiratory cells may be even more sensitive than eia-based methods. , the increasing use of rapid tests has facilitated the assessment of rsv in tropical areas. ideally, a combination of a rapid method with viral isolation should be used for maximal rsv detection, but the cost may still be prohibitive for the meager resources available in some tropical areas. detection of rsv rna by conventional rt-pcr has shown suboptimal sensitivity, especially when compared with easy-to-perform, more sensitive rapid methods. however, more recently developed assays based on real-time rt-pcr are proving to be more sensitive than conventional rt-pcr assays, with the added conveniences of being rapid, quantitative, and amenable to simultaneous detection and subtyping of rsv directly from clinical specimens. rsv serology has limited value for case management but may be useful for epidemiologic surveys. treatment uri caused by rsv requires no specific treatment, and antibiotics are needed only when bacterial otitis media or sinusitis are present. the supportive treatment of infants with rsv bronchiolitis consists basically in preventing hypoxemia and electrolyte imbalance, in addition to aerosolized bronchodilators. the lack of obvious correlation between radiologic findings and disease severity suggests that a chest film should be recommended only for severely ill or deteriorating infants. to prevent hypoxemia, requirements may vary from simple removal of respiratory secretions and proper positioning of the infant to mechanical respiratory assistance and even extracorporeal membrane oxygenation (ecmo). pulse oximetry has been advocated to assess oxygen needs, but in tropical developing areas, where oximeters may not be available, serial clinical assessment is essential to monitor disease progression. for this purpose, crackles and cyanosis seem to correlate better with hypoxemia than tachypnea and intercostal retraction. correction of hypoxemia can be accomplished with % or lower oxygen concentrations. oxygen should be humidified with saline and delivered by mask if head boxes or tents are unavailable. the role of corticosteroids remains unclear with some evidence that they are not beneficial. the only antiviral drug currently approved for the treatment of infants with rsv is the synthetic nucleoside ribavirin, delivered by small-particle aerosol via a mist tent, mask, oxygen hood, or ventilator. it is recommended only for infants and young children with an underlying condition, such as congenital heart disease, cystic fibrosis, or immunosuppression. premature infants, infants younger than weeks of age, and severely ill infants may also be considered for therapy. aerosolized ribavirin is well tolerated, but it is expensive and its prolonged administration requires facilities that may not be available in impoverished tropical areas. passive immunotherapy with rsv immunoglobulin, in combination with aerosolized ribavirin, improved the outcome of rsv pneumonia in bone marrow transplant patients. the use of rsvintravenous immunoglobulin (ivig) or humanized monoclonal antibody against rsv has shown no benefit for the treatment of rsv infections in infants. no vaccine is currently available for rsv prophylaxis. the disease enhancement caused by formalin-inactivated vaccine in the s plus results of more recent unsuccessful trials of live-attenuated vaccines, have significantly slowed progress toward an rsv vaccine. purified fusion protein vaccine has been tested for safety and immunogenicity in seropositive children older than months, and was associated with reduction of lower respiratory tract illness, but not of rsv infection rates, in children with cystic fibrosis. these and other candidate subunit vaccines, as well as intranasal liveattenuated vaccines, should be tested in high-risk children with underlying bronchopulmonary diseases. passive immunization of high-risk infants with monthly infusions of rsv immunoglobulin during the rsv season reduced the incidence and severity of rsv infections in highrisk children. this costly intervention is the only available means of protecting high-risk children against serious rsv lri. monthly intramuscular injections of humanized monoclonal antibody should be considered for passive immunoprophylaxis during rsv season for high-risk infants such as preterm infants less than months old, children with congenital heart disease, and children less than years of age with bronchopulmonary dysplasia. hospitalized infants with rsv infection should be isolated or grouped to prevent cross-infection. hand washing; use of eye-nose goggles, gowns, and gloves; and decontamination of surfaces and fomites are additional nosocomial infection control measures. human parainfluenza viruses (hpivs) are the single most frequent cause of croup in infants and children worldwide and are second only to rsv as cause of lri in infants. , little is known about the epidemiology of hpivs in tropical countries, but these viruses have been detected in up to % of children in hospital-based ari studies in developing countries. , hpivs are distributed in two genera of the family paramyxoviridae, sharing the structural and biological characteristics already mentioned in the rsv section. hpivs are classified antigenically into types to , and hpiv- has subtypes a and b. hpiv types and are classified in the genus respirovirus, while hpiv types and are in the genus rubulavirus. hpiv- and - are the types most frequently associated with lri in children, the immunocompromised, the chronically ill, and the elderly, whereas piv- causes mostly uri in both children and adults. binding of hpiv to sialic acid in the cell membrane is mediated by the glycoprotein hn, which contains hemagglutinin and neuraminidase activities. fusion of viral and cell membranes is mediated by the viral f protein, which is cleaved by cellular proteolytic enzymes. once inside the cell, the cycle is similar to other paramyxoviridae, as summarized in the rsv section. hpivs can be propagated in primary simian or human kidney cells and in several cell lines, such as hep- , vero, mdck, llc-mk , bhk, and hela. a variety of experimental animals undergo asymptomatic infection with piv, but only higher primates develop symptoms. , epidemiology primary hpiv infection occurs early in childhood, and by age virtually all children are seropositive. an estimated one third of all viral lris in children in the united states are caused by hpiv- and - . , in most temperate regions, hpiv- and - cause epidemics in the fall of alternate years, either in co-circulation or alternating with one another. the biennial pattern of hpiv- is found in both hemispheres. hpiv- causes most croup epidemics, whereas hpiv- more frequently causes illness with milder manifestations, although it can also cause croup. hpiv- occurs endemically throughout the year, with sporadic spring outbreaks mainly among infants, and hpiv- occurs sporadically throughout the year in children and adults. , , in tropical areas hpivs may account for up to % of child hospital admissions due to lri. community-based ari studies in children under age years show higher hpiv activity during rainy seasons in tropical countries. , hpivs were the most frequent viruses detected in school-aged children with bronchial asthma exacerbations in urban nigeria. hpivs spread mainly within families and closed communities, such as nurseries, day-care centers, and pediatric wards, with high secondary attack rates. in a longitudinal study conducted with children less than years of age with ari in a day-care center for low-income families in northeast brazil, hpivs represented % of the viruses detected. the virus does not persist long in the environment and is transmitted mainly by large droplets and fomites. viral shedding usually lasts to days, but shedding of hpiv for months has been reported in very young children and immunosuppressed hosts. primary hpiv infection may cause rhinitis, pharyngitis, laryngotracheobronchitis (croup), bronchiolitis, or pneumonia. approximately two thirds of all piv infections in children result in febrile uri with associated otitis media in % to %. the remaining one third of piv infections are cases of croup, bronchiolitis, or pneumonia. , hpivs, mainly of types and , cause up to % of all cases of croup. croup is the most striking clinical presentation of hpiv infection and is most common between the ages of and months. croup is manifested by inspiratory stridor, barking cough, and hoarseness caused by subglottic edema, preceded by rhinorrhea, mild cough, and low-grade fever. , most children recover in to days, but some may develop bronchiolitis and pneumonia and present with a bronchopneumonia-croup syndrome. , , since immunity to hpivs is incomplete, infections tend to occur throughout life, but little is known about hpiv infections in adults. in general, adults have only nonspecific uri, commonly with hoarseness. hpivs can cause particularly severe diseases in immunocompromised hosts, especially children with severe combined immunodeficiency and bone marrow transplant patients. mortality in bone marrow transplant patients with hpiv infection varies from % to % in most series. , hpivs replicate in ciliated epithelial cells, causing cytolysis of the respiratory mucosa. the infection begins in the upper respiratory tract and tends to disseminate down the respiratory tree. the larynx and trachea are mostly involved in the croup syndrome, and extensive involvement of the lower respiratory tree may be present in tracheobronchitis, bronchopneumonia, and bronchiolitis. , , similar to influenza, factors determining the extent of hpiv infection include the susceptibility of the viral f protein to be cleaved and tissue-specific differences in the production of proteases to cleave it. host immunity is largely mediated by humoral immunity to the two surface proteins hn and f. virtually all children by the age of years will have seroconverted to hpivs, generally first to hpiv- but later also to hpiv- and - . at school age, a significant proportion of children will have seroconverted also to hpiv- . secretory antibody targeted to the hn glycoprotein is the best marker of protection against piv, but the protection conferred by antibodies is limited, and repeated infections will develop. t-cell immune response seems to be involved in the clearance of virus and additionally in the development of inflammatory infiltrate, edema, and excess mucus secretion, and immunocompromised hosts may develop progressive and even lethal disease. like rsv, pivs cause mononuclear interstitial infiltrate, epithelial necrosis, inflammatory exudate into the alveoli, and hyaline membrane formation in the lungs. piv is present in respiratory secretions until about days from the onset of symptoms and can be isolated in monkey kidney primary cells and several continuous cell lines. virus can be detected in the monolayers by hemadsorption with guinea pig erythrocytes in around days after inoculation and confirmed by if. , shell-vial assays have been developed for hpiv detection but with mixed results. if of exfoliated respiratory epithelial cells has produced conflicting and sometimes disappointing results, with most studies reporting sensitivities between % and % at best. detection of viral rna by rt-pcr, including commercially available multiplex assays for several respiratory viruses, has enhanced the sensitivity of detection of hpiv from clinical samples. , real-time pcr for respiratory viruses in multiplex format is sensitive and specific for hpiv. at present, only supportive and symptomatic treatment is available for piv infections. management of croup includes supplemental oxygen and racemic epinephrine nebulization in hospitalized patients. mist therapy, although traditional, has no proven value. short-term, high-dose systemic corticosteroids may reduce the need for intubation, and nebulized budesonide has a rapid effect and is as safe and efficacious as nebulized epinephrine in moderately severe croup. several antiviral agents have in vitro activity against hpivs, but none has reached clinical testing. there have been anecdotal reports of reduced hpiv shedding in immunocompromised patients treated with ribavirin, but this finding has not resisted scrutiny. future possibilities include the bcx and bcx compounds, whose design is based on the threedimensional structure of the hn protein, which inhibit the hemagglutinin and neuraminidase activities of the protein and were effective in vitro and in an animal model against hpiv- , - and - . no interventions are available for the prevention of hpiv infections. early trials with inactivated hpiv vaccine in the s were unsuccessful. recently, a live-attenuated, coldadapted hpiv- vaccine was found to be immunogenic for children as young as month of age and holds promise for further development. the same vaccine was tested in combination with a live-attenuated rsv vaccine candidate, showing that this approach is feasible and deserves further study. characterization of hpiv proteins hn and f has led to development of subunit immunogens that showed efficacy in animal models. human rhinoviruses (hrvs) are the most frequent respiratory pathogens of humans. they were the most frequently isolated viruses in children under years of age with ari in an urban slum in northeast brazil. human rhinoviruses are small, nonenveloped, positivestrand rna viruses in the family picornaviridae, with over identified serotypes. hrv serotypes have been classified according to receptorspecificity into three groups. the major group includes serotypes whose receptor is intercellular adhesion molecule- (icam- ); the minor group contains serotypes whose receptor is the low-density lipoprotein receptor (ldlr); and the remaining serotype, hrv- , utilizes a sialoprotein as cell receptor. unlike other picornaviruses, hrvs are acid-labile, a property that distinguishes them from enteroviruses. the hrv genome is a monocistronic single-stranded rna, packed in an icosahedral capsid composed of pentamers. surrounding the fivefold vertex, each pentamer contains a . -to . -nm-wide canyon that contains the receptor binding site. following receptor binding, the viral positivestrand rna is released into the cytoplasm and directs the synthesis of a polyprotein, whose cleavage products include an rna polymerase. this enzyme will produce an expanding pool of positive-strand rna using as a template an intermediate negative-strand rna. the positive-strand rna can be either translated into virion proteins or packaged as a genome into newly assembled virions. the hrv replication cycle takes place in the cytoplasm, and mature virions are released when the host cell is lysed. hrvs are resistant to ethanol, ether, chloroform, and nonionic detergent but are sensitive to uv light; to ph lower than and higher than ; and to halogens such as chlorine, bromine, iodine, and phenolic disinfectants. they are stable for days on environmental surfaces and for years at minus °c. hrv infects only higher primates and causes illness only in humans. several cell lines of primate origin support hrv propagation, but certain strains of hela cells and human embryonic fibroblasts provide higher sensitivity for hrv isolation from clinical specimens. the optimal growth temperature for hrv is °c to °c. hrv infections occur in people from all continents, including remotely located population groups, such as bushmen from the kalahari desert, native alaskans, and an isolated amazon indian tribe. hrv has been estimated to cause up to % of all autumn colds in temperate climates. in tropical countries, very few community-based studies of viral ari have used adequate hrv detection methods, and this has limited the assessment of the actual impact of hrv in those areas. however, available evidence indicates that hrv is frequently associated with ari in children in brazil. in fortaleza, a city in northeast brazil, hrv detected by isolation in cell culture represented % of the viruses in children under years of age with ari. in salvador, another city in the same region, hrv represented % of the viruses detected by rt-pcr in association with ari in children younger than years of age attending a day-care center for the underprivileged. data on the frequency of hrv among adults in tropical countries are even more scarce. in singapore, hrv was detected in % of the samples obtained from adults with ari symptoms attending primary care centers. hrv transmission requires close exposure and occurs mainly by hand-to-hand contact, followed by self-inoculation into the eye or nose, but also by airborne spread. once hrv reaches the nasal cavity, infection occurs in virtually % of susceptible subjects, and approximately % of those infected develop illness after a -to -day incubation. children play a central role in spreading the virus in the household. evidence suggests that indoor hrv transmission is favored by high relative humidity and crowding of young children, as occurs in the united states at the beginning of the school term, which may explain the autumn seasonal peak of hrv. in tropical northeastern brazil, however, where relative humidity remains above % reaching % during the rainy season, longitudinal studies have found no obvious hrv seasonality. , hrv colds are indistinguishable from colds of other viral causes and consist of nasal discharge, nasal obstruction, sneezing, sore or scratchy throat, hoarseness, cough, and headache. facial and ear pressure may be present. fever and malaise are uncommon. these symptoms last approximately days but may persist for up to weeks in % of cases. infants and toddlers may display only nasal discharge and be otherwise asymptomatic. the majority of patients have obstruction and mucosal abnormalities of the sinus cavities, eustachian tubes, and middle ear, which predispose to secondary bacterial sinusitis and otitis media, each complication found in approximately % of all colds. hrv rna may be detected by rt-pcr in maxillary sinus brushings in % of adults presenting with acute sinusitis, and in % of the samples of middle ear fluid from children less than years of age with diagnosis of acute otitis media. hrv is frequently associated with exacerbations of chronic obstructive pulmonary disease and asthma attacks in children over years of age and in adults. , , hrv replication is restricted to the respiratory epithelium, taking place in scattered ciliated cells of the nose and in nonciliated cells of the nasopharynx. this tropism seems to be a consequence of receptor availability. infection of a limited number of cells triggers the release of cytokines, chemokines, and inflammatory mediators, which together with stimulation of the local parasympathetic nerve endings, results in the cold symptoms. kinins, prostaglandins, and proinflammatory cytokines and chemokines may contribute to vasodilation, increased vascular permeability, influx of polymorphonuclear leukocytes, exocrine gland secretion, and nerve ending stimulation, resulting in nasal obstruction, rhinorrhea, sneezing, cough, and sore throat. serotype-specific neutralizing igm, igg, and iga antibodies develop in most infected persons in to days and persist for years. protection from infection is partially attributed to the presence of iga antibody in nasal secretions, and recovery from illness is more dependent on cell-mediated immunity. hrv-induced blastogenesis, natural killer cell activity, mitogen-stimulated cell production of il- and ifn-γ have been documented during hrv infection. , hrv induces the expression of human β-defensin (hbd- ) in the respiratory epithelium, which supports a role for hbd- in host defense to hrv infection. hrv can be detected in respiratory secretions by isolation in cultures of susceptible cell lines. hrv shedding peaks around hours after infection and declines rapidly, but may remain at low levels for up to weeks. cultures should be kept at °c to °c in a roller drum and examined for to days. the presence of hrv, indicated by the typical cpe, is confirmed by the acid sensitivity of the isolate. rapid immunocytochemical methods are not available because of the large number of serotypes. rt-pcr in clinical samples is more sensitive and less tedious than hrv isolation, and the recently introduced real-time pcr-based assay is more sensitive than conventional rt-pcr. pcr-based assays have been useful in studies to assess the impact of hrv in different settings. the homotypic nature of hrv antibodies restricts serology to experimental settings. trials of antiviral agents for hrv have been conducted, but no specific treatment suitable for routine use has yet been identified, mainly because of lack of potency, untoward side effects, and drug delivery problems. ruprintrivir, a selective inhibitor of hrv c protease, has potent, broad-spectrum anti-hrv activity in vitro. a double-blind, placebo-controlled clinical trial of intranasal ruprintrivir in experimental hrv infection reduced symptoms by % and also decreased viral titers and nasal discharge. symptomatic relief from cold symptoms can be obtained with a broad variety of nonprescription medications. systemic sympathomimetic decongestants, such as pseudoephedrine, may reduce nasal obstruction, first-generation antihistamines may reduce sneezing and rhinorrhea, and nonsteroidal antiinflammatory drugs such as naproxen or ibuprofen may reduce headache, cough, and systemic symptoms. the large number of hrv serotypes with minimal crossantigenicity has hampered the development of an hrv vaccine. it may be possible to reduce exposure to hrv by hand washing after contact with a cold sufferer or after handling objects that may have been contaminated with respiratory secretions. studies in experimentally infected volunteers show that application of the virucidal agents salicylic acid or pyroglutamic acid to the hands reduced recovery of rhinovirus from the hand skin of treated persons as compared with controls. this result suggests that rhinovirus transmission can be prevented by virucidal hand treatments. short-term, postexposure prophylaxis by intranasal ifn-α significantly reduced the incidence of hrv colds in household contacts of an index case. however, the cost and difficulty of making the drug available to homes in a timely fashion reduce the utility of this approach for extended use by populations, especially in tropical countries. ruprintrivir has also been evaluated for prophylaxis of hrv colds starting hours prior to inoculation of human volunteers. this approach reduced the proportion of subjects with positive viral cultures and viral titers but did not affect the frequency of colds. respiratory infections caused by adenoviruses are among the most frequent illnesses that these viruses cause, particularly in children under age years. adenoviruses have been frequently isolated in ari studies in tropical countries. in the south cone of south america, adenoviruses were the second most frequent virus recovered from children hospitalized for ari. adenoviruses are nonenveloped, icosahedral dna viruses of the genus mastadenovirus in the family adenoviridae. adenoviruses are distinguished antigenically by group-specific (a through f) and type-specific ( through ) antigens and by genomic subtypes identified by restriction site mapping. , the adenovirus capsid consists of three morphologically, antigenically, and functionally distinct types of capsomers: hexons, penton bases, and fibers that project from the penton bases. the hexon and penton bases contain complementfixing, group-specific antigens common to all human adenoviruses, whereas the fibers have primarily neutralizing and hemagglutination-inhibiting, type-specific antigens. adenoviruses are commonly accompanied by small, singlestranded dna parvoviruses known as adenoassociated viruses, which do not seem to cause any specific disease. most people have antibodies to at least one of the four serotypes of adenoassociated virus by age years. the fiber protein binds to the host cell, through the protein coxsackie and adenovirus receptor (car) of the immunoglobulin superfamily, which serves as a high-affinity receptor for adenoviruses. the class i major histocompatibility complex (mhc) also may serve as receptor for adenovirus . ligand-receptor interaction facilitates interaction of the penton base with cell surface integrins, which triggers entry. after endocytosis, the double-stranded linear genomic dna is transported to the nucleus, where "early" and "late" sets of viral genes are transcribed, resulting in mrnas coding for structural and nonstructural proteins. virus assembly takes place in the nucleus, and the infectious cycle is completed by the release of up to million virions upon cell lysis. adenoviruses replicate well in continuous cell lines of epithelial origin, such as hep- , hela, and a , and can be adapted to grow in human embryonic lung fibroblasts. they are stable over a wide ph range ( to ), resistant to isopropyl alcohol, ether, and chloroform, stable for weeks at room temperature and for years at approximately °c or colder, and can be lyophilized. they are inactivated by sodium hypochlorite and a temperature of °c for minutes. respiratory transmission of adenoviruses occurs at all ages but is of prime importance during epidemics among military recruits. ocular transmission has been associated with swimming pools and physician offices where sterilization or hand washing has been inadequate. asymptomatic infection and a prolonged carrier state are common. low-number adenovirus serotypes ( , , , and ) are more frequent before age years and account for % to % of cases of uri and approximately % of cases of lri in children. in adults, adenoviruses occur sporadically and cause mostly uri. infections by adenoviruses types and are usually epidemic, with attack rates of % to % per week in newly assembled military recruits, whose adenovirus carriage rate may be as high as %. in this group, the adenoviral syndromes vary from mild colds to severe lri, but overall attack rates may reach %, with % to % of the individuals needing hospitalization. in temperate climates, adenoviral infections are more frequent in late winter, spring, and early summer, whereas in northeast brazil they seem to occur year-round. in salvador, also in northeast brazil, adenoviruses were detected in % of children younger than age years with ari in a day-care center. in tropical areas the incidence of adenovirus infections in military recruits is lower, and different serotypes may be involved. pharyngoconjunctival fever, commonly caused by adenoviruses types and , may be epidemic or endemic among children during the summer in temperate climates. inadequate chlorination or filtration of swimming pools and lakes has been associated with epidemics. the incubation period of adenovirus infections averages days. adenovirus respiratory diseases may involve all parts of the respiratory tract, and up to % of nonepidemic infections are asymptomatic. in fact, adenoviruses were discovered because of their propensity for latency in adenoidal tissue. , in southeast brazil, adenoviruses were detected with equal frequency in wheezing young children and asymptomatic controls. most adenoviral illnesses consist of febrile colds, and in children the fever may be high and long-lasting. pharyngitis is common and may be associated with fever, pharyngeal exudate, granular appearance of the mucosa, and anterior cervical adenopathy, similarly to streptococcal pharyngitis. adenoviruses can be recovered from up to % of cases of pharyngitis in small children. pharyngitis may be concurrent with pharyngoconjunctival fever, a syndrome caused by adenovirus types and and characterized by conjunctivitis, frequently unilateral, which may last for to weeks, preauricular adenopathy, cough, rhinitis, malaise, and fever. the most frequent complication of adenoviral colds is acute otitis media, which occurs in up to % of cases. adenovirus lris consist mainly of bronchitis and pneumonia, and may make up over % of childhood lris in temperate areas. adenoviruses may cause permanent lung parenchymal damage, especially when concurrent with measles. epidemic adenoviral infections in military recruits have a spectrum of clinical manifestation ranging from colds to severe pneumonia. typically, however, the manifestations are fever, pharyngeal symptoms, cough, chest pain, headache, and malaise. overwhelming pneumonitis may be part of disseminated adenoviral infections in newborn infants and patients with immunodeficiencies, including acquired immunodeficiency syndrome (aids). however, the frequent concomitance of other respiratory pathogens in aids patients and the high prevalence of asymptomatic adenovirus infection shed doubt on the causal role of the adenovirus in these patients. adenoviruses are also an important cause of epidemic keratoconjunctivitis. , while most adenoviral aris are self-limited and uncommonly associated with death or permanent sequelae, adenoviruses alone or associated with other pathogens have been recovered from % of fatal cases of lri in argentina. adenoviral respiratory disease results from necrosis of cells of airway epithelia, and viremia may result in disseminated infection in immunocompromised persons. bronchiolitis, interstitial pneumonitis, and mononuclear cell infiltrates are part of the inflammatory process in the lungs. it remains unclear why certain strains are more virulent than others. for example, the genomic variant b h was associated with the majority of fatal lower respiratory disease in south america. in addition to lytic infection, adenoviruses may become latent in epithelial and lymphoid cells, which is probably important to maintaining the virus in populations. a possible role of latent adenovirus in the pathogenesis of chronic airway inflammation has been suggested. , , protection from adenovirus infection and disease is mainly due to type-specific neutralizing antibody, but reinfections, mostly asymptomatic, may occur. a long-lived t-cell immune response develops in most infected immunocompetent persons and is not only responsible for recovery but also is involved in tissue pathologic changes. adenoviruses can be detected in respiratory, ocular, or ear secretions, but clinical correlation is required, because asymptomatic virus shedding is common. isolation of adenoviruses in cell culture with identification by if constitutes the standard diagnostic method, but direct detection of viral antigens or viral dna by pcr in clinical samples is an attractive rapid alternative. rapid antigen detection by immunochromatography is around % sensitive in comparison with cell culture, and easily can be used in point-of-care diagnosis of adenovirus. however, both conventional and real-time pcr are more sensitive than cell culture is. positive results by pcr should be interpreted with caution, given the propensity of adenoviruses to cause latency. adenoviruses cause a characteristic cpe in a variety of cell lines of human origin, and maintenance of cultures for weeks combined with blind passage (i.e., passage of cells even without obvious cpe to see if it develops after passage) may increase adenovirus recovery. , inoculation of cells by centrifugation followed by immunostaining may shorten the detection time. several serologic tests can detect antibodies to the common hexon antigen. however, their clinical utility is restricted. at present, there is no routine effective antiviral treatment for adenovirus infections. successful therapy of severe adenoviral infections in immunocompromised patients with iv ribavirin has been reported. cidofovir has shown some efficacy in the rabbit ocular model of adenoviral infection. iododeoxyuridine and adenine arabinoside were unsuccessful in the treatment of adenoviral keratoconjunctivitis. a live vaccine consisting of wild-type adenovirus packaged in enteric-coated capsules induces immunity by ensuring enteric infection without infection of the respiratory tree. this approach has been used successfully to vaccinate military recruits against adenoviruses types and . proper sterilization, hand washing, and chlorination can prevent adenovirus spread via tonometers, hands, and swimming pools. coronaviruses are enveloped viruses with distinct virion morphology, displaying widely spaced, long petal-shaped spikes at the surface, that confer a crownlike appearance, the origin of the name corona. the envelope contains a long helical nucleocapsid with single, positive-stranded rna, to kb in size, which is the largest known viral rna genome. until very recently, only three human coronaviruses (hcovs) were known to exist: hcov- e, hcov-oc , and the cov associated with severe acute respiratory syndrome (sars-cov). recently, two groups in the netherlands almost simultaneously published studies that resulted in the identification of two new strains of hcov: hcov-nl and hcov-nl. in addition, pcr primers directed to conserved replicase a sequences of animal covs led to the identification of yet another hcov detected in . % of children from new haven, connecticut with symptoms of ari. this agent was designated hcov-nh and is likely to represent the same species of hcov-nl and -nl . on the basis of antigenic and genetic studies, the known human coronaviruses are distributed in three of the four coronavirus groups so far identified. hcov- e, -nh, -nl, and -nl belong to group i, hcov-oc belongs to group ii, and sars-cov is the only known constituent of group iv, while group iii contains no known human viruses and consists only of the avian infectious bronchitis virus. coronavirus rna synthesis occurs in the cytoplasm via a negative-strand rna intermediate. the viral rna possesses a ′ cap followed by a leader sequence and an untranslated region, with another ′ terminal untranslated region followed by a poly(a) tail. the genome is polycistronic and the synthesis of subgenomic negative-sense rnas is done by discontinuous transcription to originate a nested set of subgenomic mrnas that share the ′ leader sequence and overlap at the ′ end. the envelope contains the structural proteins s (spike), m (membrane), e (envelope), and only in the case of some group ii coronaviruses, ha (hemagglutinin). the s glycoprotein contains neutralizing and t-cell epitopes and functions as the cell receptor ligand, thereby determining tissue tropism. the m protein is embedded in the envelope and interacts with the n (nucleocapsid) protein during maturation. in addition to the nucleocapsid and envelope proteins, a replicase is present in cells infected by all coronaviruses. new virions assemble by budding through intracellular membranes and are released through vesicles of the secretory pathway. hcov- e and -oc are considered to be second only to rhinoviruses as agents of common colds, causing infections with variable frequency, depending mainly on the detection method and season of the study. up to % of mild upper respiratory tract infections in adults have been attributed to hcov- e. while hcov- e and -oc are documented causes of colds in temperate regions, their impact as causes of respiratory infections in tropical regions has not been defined. human coronaviruses were first isolated in england, almost years ago, in human organ cultures of tracheal and nasal tissues. there have been relatively few field studies based on hcov isolation in cell culture, likely because these viruses are too fastidious to be propagated, but most respiratory isolates obtained so far have been antigenically similar to either hcov- e or -oc . these agents have the same structural features as the other members of the family. the s protein of hcov- e binds to the metalloprotease human aminopeptidase n at the cell surface, and entry is independent of enzymatic activity of the receptor. the hemagglutinin of hcov-oc binds to sialic acid present in glycoproteins on the cell surface and this interaction facilitates infection, but to the best of our knowledge, a specific receptor has not been identified for this agent. hcovs have been found throughout the world and are considered to be the second most frequent cause of common cold, accounting for an average rate of % of respiratory illnesses in the general population in the united states. however, the rates may be quite variable from year to year, ranging from % to % in years of peak activity. hcov infections occur mainly in the winter and spring months, but summer activity has also been documented. during the autumn peak of rhinovirus activity, % of the adults with a cold negative for rhinovirus were positive for hcov by rt-pcr in charlottesville, va. hcov- e has caused well-documented winter outbreaks at -to -year intervals in temperate regions. , similarly, winter outbreak of hcov-oc has also been detected in europe. in contrast, little is known about the prevalence of hcov- e and -oc in tropical countries. in brazil, the activity of hcov- e as cause of respiratory infections in nonhospitalized children was first documented by serology in the early s, with a seropositivity rate of % in adults by complement fixation assay. the usual manifestations of hcov infection are typical common colds. the incubation period tends to be day longer that that for rhinovirus colds, with illness duration of to days. low-grade fever may occur in up to % of the patients, and in addition to nasal symptoms, cough and sore throat occur frequently. more serious infections of the lower respiratory tract caused by hcov have also been documented, either sporadically in infants with pneumonia and immunocompromised patients, or in up to % of previously healthy marine corps recruits with pneumonia. , in addition, hcov- e and -oc have been recognized in association with influenza-like illnesses in frail elderly patients. eight of ( %) nasopharyngeal swabs from older patients hospitalized for cardiopulmonary illnesses during the influenza seasonal outbreak in rochester, n.y., were positive for hcov (five for hcov- e). respiratory hcov infections have been associated with exacerbations of asthma, chronic bronchitis, and recurrent wheezing in children. , hcov was detected by rt-pcr in of ( %) episodes of asthma in children to years old in england. in brazil, hcov was detected in respiratory samples from ( oc and e) of ( %) children younger than years of age who came to the er with wheezing. similarly to hrv, hcov infections have been frequently recognized in association with otitis media and maxillary sinusitis in children and adults. hcov was detected by rt-pcr in the middle ear effusion or nasopharyngeal aspirate from of ( %) children with acute otitis media in finland and in nasal swabs from of adults with acute maxillary sinusitis. there is no convenient small animal model to study the pathogenesis of hcov, and humans naturally or experimentally infected are the only source of information obtained in vivo. hcovs are transmitted by the respiratory route, and experimentally infected volunteers shed virus for approximately days, beginning hours after infection, which is approximately the time of onset of symptoms. , the peak of symptoms occurs to days postinoculation. hcov- e is known to infect airway epithelial cells from the apical surface, where the receptor is constitutively expressed, and to exit productively infected cells through the same route. ultrastructural studies of nasal epithelium of volunteers experimentally infected with hcov- e revealed significantly greater epithelial cell damage, ciliary loss, and cytolysis in virus-inoculated subjects than in sham-inoculated ones on day postinfection. in the united states, seropositivity to hcov-oc and - e rises during the first years of life, and around % of adults are seropositive. symptomatic reinfections are possible, despite the presence of antibodies, suggesting rapidly waning immune response or circulation of closely related but antigenically different viruses. , several studies indicate that respiratory hcovs are able to reach the central nervous system. , , the recently reported temporal association between hcov-nh infection and kawaski disease awaits confirmation. laboratory diagnosis in clinical samples by isolation is tedious, because the two best characterized strains of hcov are difficult to grow in routine cell cultures. since primers can be developed for relatively constant parts of the genome, rt-pcr-based assays for hcov- e and -oc have recently become the best alternative to other methods of detection. more recently, a quantitative real-time pcr-based assay for hcovs has been developed, providing a faster means for detection and determination of viral load with potential applications in clinical studies. serologic diagnosis of hcov by eia is sensitive and specific and has been useful in epidemiologic surveys. intranasal interferon protects against experimental infection with hcov- e, but no specific antiviral therapy is available, and treatment of hcov-induced colds remains largely symptomatic. no vaccines are currently available for hcov. the hcov that fulfills koch`s postulates as the causative agent of sars shares structural features and genome organization of the family coronaviridae ( fig. - ) . the prompt recognition of the peculiar morphology of a coronavirus in the electron microscopic studies of vero e cells inoculated with oropharyngeal material from a patient was the initial finding that resulted in the identification of sars-cov. the viral genome is , nucleotides in length, with more than open reading frames coding for putative proteins, some of which have unknown functions. sars-cov is phylogenetically different and equidistant from all previously known coronaviruses, but isolates from different origins are relatively homogeneous genetically. genome analysis reveals that sars-cov is neither a host-range mutant nor a recombinant of respiratory tract viral infections ■ previously known coronaviruses but rather an independently emerged virus. sars-cov seems to have evolved from an animal sars-like virus, acquiring greater fitness in humans during the course of the outbreaks, probably through the appearance of nucleotide deletions in open reading frame . it is also noteworthy that genetic signatures present in the genomes allow for differentiation of isolates obtained from different clusters. the replicative cycle of sars-cov is thought to follow the same main steps as other coronaviruses. sars coronavirus (sars-cov) probably emerged around november in the province of guangdong, china, where there was no serologic evidence of infection caused by this virus in sera of healthy humans sampled prior to that time. at the beginning of the outbreak, many affected individuals in guangdong were directly or indirectly involved with game trade, and indeed, palm civets and raccoon dogs from wildgame markets in the area were later found to harbor a cov % homologous to sars-cov at the nucleotide level. this suggests that animal-to-human interspecies transmission was involved in the outbreak, providing the source of an agent that later adapted to efficient human-to-human transmission. , interestingly, shortly after the lifting of a wildlife trade ban that originally had been imposed to control the sars outbreak, new cases were again detected in guangdong, all of them caused by viruses newly introduced from animals. since the ban was reinstalled, there have been no further naturally acquired human cases of sars in guangdong. remarkably, . % of serum samples from adults recruited in in hong kong tested positive for sars-cov antibodies, suggesting that a small proportion of healthy people from hong kong, as opposed to guangdong, china, had been exposed to sars-related viruses at least years before the outbreak. it is probable that sars-cov precursors previously crossed the species barrier and may even have caused subclinical human infection, but perhaps only occasionally this event generated strains adapted to successful human-to-human transmission. sars-cov is mainly transmitted between humans by the deposition of infected droplets or aerosols on the respiratory epithelium. the number of confirmed secondary cases generated by one index case of sars is relatively low, ranging from . to . , suggesting relatively inefficient transmission. in addition, transmission is infrequent during the first days of illness, partly because of the low viral load in respiratory secretions during that phase. for reasons not completely understood, some sars patients, identified as superspreaders, disproportionately contribute to the generation of a high number of secondary cases. excretion of sars-cov in sputa and stools may average and days, respectively, after symptom onset, but an excretion period as prolonged as days has been documented in stools. such prolonged shedding of virus in feces raises the possibility of oral-fecal transmission and, in fact, one outbreak of sars was attributed to a faulty sewage system. case-fatality rates estimated based on cases admitted to hospital have been around % for patients younger than age and % for those older than age years. however, it is likely that case-fatality rates based on all infections occurring in the community would be lower. transmission of sars-cov among health-care workers and between patients in the hospital setting played a pivotal role in outbreak propagation. analysis of data from initial outbreaks indicates that close contact is the most important factor leading to nosocomial transmission of this agent. despite the lack of complete studies on the sensitivity of sars-cov to different environmental conditions, there have been reports of sars-cov persisting for up to days on environmental surfaces and days in diarrheal stools. the median incubation period of sars is to days. clinical symptoms and signs of sars appear to days after exposure, and systemic symptoms, such as fever, chills, myalgia, and malaise, usually appear first. respiratory symptoms appear to days later, represented most frequently by nonproductive cough, dyspnea, chest pain, headache, and sore throat. diarrhea and vomiting may occur. chest radiograms frequently reveal infiltrates consistent with viral pneumonitis, consisting mostly of consolidations and ground-glass opacifications. computed tomography (ct) scans in patients with normal or equivocal chest radiograms may show unilobar or multilobar abnormalities. fever generally subsides in hours, but one or two relapses within to days are frequently observed. lymphopenia with reduction of both cd + and cd + cells, slight decrease in platelet counts, prolonged coagulation profile, and elevated serum enzymes (lactic dehydrogenase [ldh], creatinine kinase [ck], and c-reactive protein [crp]) are often observed. around one third of patients may have cd + lymphocyte counts below cells/mm and higher susceptibility to secondary infections. watery diarrhea with an average of six evacuations per day is common. , , radiologic worsening of the pulmonary lesions seen at admission, with or without appearance of new lesions, is a frequent observation, with development of diffuse groundglass changes frequently heralding the development of acute respiratory distress syndrome (ards). hypoxemia is noted in approximately half of the patients at around days after the onset of symptoms, and a high proportion of those admitted to the intensive care unit (icu), especially older males, require mechanical ventilation around day . development of spontaneous pneumomediastinum during follow-up is not uncommon, probably as a consequence of ruptured peripheral lung lesions into the pleural space. prognosis is related to the level of viral replication in tissues, and patients with high viral loads in serum, nasopharyngeal aspirates, or feces, as well as those in whom virus can be detected from multiple sites, tend to have poor clinical outcome. in addition to old age and severe underlying diseases, ck and crp levels have been identified as predictors of poor outcome. the n-terminal portion of the spike glycoprotein is needed for virus attachment to the virus receptor, identified as the metallopeptidase angiotensin-converting enzyme homolog (ace- ), but it is unclear whether the mechanism of entry is contingent on ph-dependent endocytosis. some inconsistencies between ace- and sars-cov tissue distribution suggest that ace- may not be the only receptor, or that a coreceptor molecule may be needed for cell infection. sars-cov spike protein can also bind the dendritic cell-specific c-type lectin intercellular adhesion molecule -grabbing nonintegrin (dc-sign), which does not result in dendritic cell infection by the agent but allows for sars-cov to be transported to susceptible target cells elsewhere. sars-cov has been detected in studies using different combinations of immunohistochemistry, in situ hybridization, and electron microscopy in pneumocytes and on the apical surface of enterocytes. marked inflammatory infiltrates and mucosal atrophy have not been observed in the intestine, and the pathogenesis of the sars-cov-related diarrhea remains largely unknown. sars-cov viral load in the upper airways is low in the initial days, with the peak at day of illness. quantitative rt-pcr for sars-cov in nasopharyngeal aspirates from patients who tested positive at admission revealed viral loads around copies/ml on days and after clinical onset, and peak copies/ml on day . higher viral loads can be detected in the lower respiratory tract than in the upper airways. pulmonary tissue shows diffuse alveolar damage, mixed infiltrate, lung edema, hyaline membrane, abundant macrophages in alveoli and interstitium, and syncytia formation. besides respiratory secretions and stools, sars-cov can be detected in urine in up to % of patients, with titers averaging . copies/ml, in association with abnormal urinalysis results. the effect of sars-cov infection on the immune system is highlighted by pronounced t-cell lymphopenia and elevation of several inflammatory cytokines (il- β, il- , and il- ) and chemokines (mcp- and ip- ) observed in sars patients. while mcp- is likely to be involved in the lung monocytic/macrophagic infiltrate, its role is not firmly established, since other viral diseases that are associated with elevated mcp- , such as influenza, do not include such prominent histologic features. in addition, since immunologic markers in the peripheral blood may not reflect what happens in the microenvironment of the lung, the pathogenic importance of these findings is not clear. co-inheritance of hla-b* and -b is higher among sars patients than in the general population, favoring a role for the genetic background in susceptibility to sars-cov. the pathogenesis of the t-cell lymphopenia remains unknown. seroconversion has been documented in % of the patients at around days and the rise in igg titers correlates with decrease in viral load. paradoxically, clinical worsening also occurs during this phase, suggesting that, rather than unchecked viral replication, immunopathologic factors may be responsible for the lung lesions. while infection in experimental animals, such as cynomolgus macaques, ferrets, cats, golden syrian hamsters, mice, and african green monkeys, does not induce disease that mimics that in humans, these models are important for studies of pathogenesis and development of vaccines and therapy. in addition, the development of an infectious cdna clone of sars-cov should permit reverse genetics experiments and may help elucidate determinants of viral pathogenesis. low viral loads in the upper respiratory tract in the first few days of illness account for the relatively poor sensitivity ( % to %) of first-generation rt-pcr for diagnosis in that period. sars-cov is detectable by rt-pcr in nasopharyngeal aspirates in only one third of patients at presentation and in two thirds at day . rt-pcr may be positive for sars-cov in stools from as much as % of patients at day , and in urine in % of samples at day . testing multiple nasopharyngeal, serum, and fecal samples increases the sensitivity of the diagnosis by rt-pcr. , to overcome the low sensitivity of conventional rt-pcr, quantitative real-time pcr-based assays for sars-cov have been developed that improve sensitivity and turnaround time, allow for amplification and analysis to be done in a closed system, and thus reduce cross-contamination. in addition, the capability of the assay to quantitate viral load has contributed not only to understanding viral pathogenesis but also to predicting outcome, since high viral loads are associated with poor prognosis. the ability to grow sars-cov in vero e cell cultures was critical to identifying the agent. sars-cov can be recovered by isolation from respiratory secretions, feces, and urine in the first weeks of illness, but the overall sensitivity is relatively low and recovery is more likely to be successful from respiratory secretions than from stools and urine. recent small outbreaks of sars-cov originating in laboratories have heightened concern about laboratory safety issues regarding sars specimens. the who guidelines for biosafety in the diagnosis of sars (updates available at the who web site) recommend that propagation of sars-cov in cell culture for isolation or for preparation of viral stocks and cell slides be performed in biosafety level (bsl ) laboratories, whereas handling serum and blood specimens for routine tests and serology can be performed in bsl laboratories. nucleic acid extraction procedures, inoculation of bacterial or mycologic cultures, and preparation of sample smears can be done in bsl laboratories, observing bsl work practices (use of safety cabinets, sealed centrifuges, protective equipment, % bleach spillage decontamination, and proper waste disposal). although not useful for early diagnosis, seroconversion determined by ifa or eia remains the gold standard for confirming sars diagnosis. igg seroconversion is detectable in over % of patients at around day . antibody crossreaction with other human coronaviruses, however rare, remains a possibility; therefore, confirmation of positive serology by an independent neutralization assay should be performed if available. the main component of treatment of sars patients is supportive therapy, chiefly the management of hypoxemia and ards. during the outbreak, treatment included a broadspectrum antiviral agent (ribavirin) and immunosuppressive doses of corticosteroids, aimed at reducing the immunopathologic damage to the lungs. the use of high-dose steroid therapy is controversial and for the most part supported by anecdotal evidence, whereas the use of ribavirin is based on the broad antiviral spectrum of the drug. however, sars-cov is only modestly susceptible to ribavirin in vitro, and therapeutic doses are difficult to achieve clinically. since it became possible to grow sars-cov in culture, many potential antiviral compounds have been evaluated in vitro, but just a few have been tested in animal models and even fewer are in clinical testing. interferons (ifn-αn /n , leukocytic ifn-α, ifn-β) and hiv protease inhibitors were consistently active in vitro and may be considered for animal testing and clinical trials. the resolution of the structure of sars-cov principal protease has prompted studies of the inhibitory capacity of known anti-hiv protease inhibitors for treatment of sars. in one open-label study, a combination of hiv protease inhibitor (lopinavir plus pharmacokinetic booster ritonavir) and ribavirin was used to treat sars patients and the outcomes were compared with historical controls treated with ribavirin alone. at day after onset of symptoms, development of ards or death was significantly less frequent in the group treated with the combination ( . %) than in historical controls ( . %). in addition, peak viral loads in respiratory samples and stools were reduced in the group treated with the combination as compared with controls. however, since there were differences in outcome predictors, such as sex, platelet counts, and ldh levels, between the two groups, these results should be interpreted with caution. a preliminary open-label study found that a restricted number of patients treated with subcutaneous interferon alfacon- in association with corticosteroids showed reduced oxygen-saturation impairment and faster resolution of radiographic chest findings than those treated with corticosteroids alone. convalescent plasma has also been tested in the treatment of sars patients. in one preliminary uncontrolled study, convalescent plasma may have reduced the frequency of poor outcome when given before days of illness. it is impossible to predict whether naturally reemerging sars-cov would be likely to cause a global outbreak. nevertheless, a vaccine for this agent would be relevant for high-risk individuals, such as workers in laboratories, hospitals, and game-animal farming. therefore, considerable effort has been directed at developing such a vaccine. it has been shown that sars-cov spike protein produced in bacteria and expressed on chimeric parainfluenza virus, as well as spike protein-encoding dna, induced neutralizing antibodies and protected experimental animals from challenge with live virus. at present, no sars-cov vaccine is available for human use. therefore, in the absence of person-to-person transmission of sars-cov worldwide, prevention of future outbreaks of sars requires careful surveillance. the goal is to maximize early detection of new cases of sars to implement control measures, thereby minimizing social disruption. to reach this goal, the cdc recommends testing for sars-cov in patients who require hospitalization for radiographically confirmed pneumonia or ards without identifiable etiology and who have one of the following risk factors in the days before the onset of illness: ( ) travel to mainland china, hong kong, or taiwan, or close contact with an ill person with a history of recent travel to one of these areas, or ( ) employment in an occupation associated with a risk for sars-cov exposure (e.g., health-care worker with direct patient contact; worker in a laboratory that contains live sars-cov), or ( ) belonging to a cluster of cases of atypical pneumonia without an alternative diagnosis (updates on these recommendations are made available at the cdc web site http://www.cdc.gov/ncidod/sars). during times of overt sars activity, prevention of humanto-human transmission is pivotal to curtailing outbreaks. although sars infectiousness relative to the onset and termination of clinical symptoms has not been accurately determined, it is clear that shortening the time from onset to hospital admission and isolation reduces the risk of transmission, thus contributing substantially to curtailing of outbreaks. identification of new cases through contact tracing played an important role in the control of the outbreaks registered so far. stringent isolation procedures must be adopted for confirmed and suspected cases, which require a high level of alertness among health-care workers for early identification of sars cases. the scenario may be further complicated in situations in which other diseases such as influenza and hantavirus pulmonary infections may occur simultaneously. rates of transmission of sars-cov among health-care workers vary, depending on stringency of control measures adopted, presence of so-called superspreaders in the hospital, and kind of activities carried out by personnel, especially as related to proximity to the index case. assisting during intubation, suctioning, and manipulating ventilatory apparatuses seem to be high-risk activities. while studies conducted in different settings have produced conflicting results, one study in toronto found that up to % of the nurses who cared for sars patients in critical care units became infected. the presence of severe watery diarrhea may add to the challenge for the infection control team. an updated set of recommendations for health-care and laboratory personnel is available at the cdc web site (http://www.cdc.gov/ncidod/sars). a new paramyxovirus was described in the netherlands in , in association with respiratory illness in children. the agent was first detected by analysis of previously unidentifiable viral isolates that induced cytopathic effect in llc-mk cell cultures. the isolates were recovered over a -year period in respiratory secretions from children with ari occurring in the winter time. electron microscopy of cell culture isolates revealed paramyxovirus-like particles, and rna sequencing revealed genome sequences and organization consistent with a paramyxovirus of the subfamily pneumovirinae, most closely related to avian pneumovirus of the genus metapneumovirus. rather than an avian virus that can also infect humans, this agent is now recognized as a primarily human pathogen, and thus has been named human metapneumovirus (hmpv). , hmpv antibodies detected in sera collected in in the netherlands indicate that this agent has been in circulation for at least to decades. agent hmpv particles are enveloped, pleomorphic, spherical, and filamentous particles, with a mean diameter of about nm. , complete genome sequences of hmpv are available and, in contrast to the genomic organization of pneumoviruses, metapneumoviruses have different positioning of the genes between m and l and lack ns and ns genes. , similar to hrsv, genetic and antigenic studies indicate that hmpv isolates cluster into two main serotype named a and b, with n gene sequences % to % similar at the nucleotide level, each subgroup including two genetic lineages (a , a , b , and b ). , , both are globally distributed. there have been no detailed studies of the hmpv replication cycle, but it is likely to be similar to that of other human paramyxoviruses. hmpv is a frequent cause of community-acquired ari in children and adults in all continents, although with variable incidence in different settings. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the united states, hmpv has been reported in up to % of lower respiratory tract illnesses whose etiology would have been unidentifiable prior to the development of assays for the detection of hmpv. in canada, during the - winter season, hmpv was detected in % of patients of all age groups from four different provinces. similar to hrsv, hmpv infections are more frequent in the colder months in temperate regions, and different strains of both subgroups a and b cocirculate during the same year. , however, only limited knowledge is available about hmpv seasonality in more tropical climates. peaks of hmpv activity have been documented in the spring/summer in hong kong, while in south africa hmpv has been detected in % to % of children with ari admitted to hospitals in the winter season. , hmpv was detected alone or simultaneously with rsv in % of children younger than years of age admitted to health-care facilities in aracaju, northeast brazil, in the months of april and may, . interestingly, hmpv was not detected by the same methods in that same city, in the following year. this apparent variability in hmpv incidence from year to year has also been observed in studies conducted in argentina and italy, where hmpv frequencies varied from % to % in three consecutive annual respiratory virus seasons. long-term prospective studies will be needed to establish whether there is a seasonal pattern in hmpv circulation in tropical regions of the world. clinically, hmpv infections resemble closely those caused by hrsv, ranging from mild upper ari to severe bronchiolitis and pneumonia. the median age of children hospitalized with hmpv infection is older than those with hrsv. hrsv in hospitalized infants and young children may require intensive care and mechanical ventilation, , , and dual infection with hmpv and hrsv appears to increase the likelihood of severe illness. , the most frequent symptoms in all age groups are fever, dyspnea, cough, wheezing/stridor, rhinitis, and sore throat. , all infected children in one study had either pneumonia or bronchiolitis, frequently accompanied by otitis media. hmpv may cause more serious infections in patients with comorbid or immunosuppressive conditions, as well as in the very young and the elderly. , in one study, all individuals older than with lower respiratory infection caused by hmpv had at least one underlying chronic or debilitating condition, including lymphoma, leukemia, or neurologic or cardiovascular diseases. hmpv infection in adults may present as influenza-like illness, acute bronchitis, or common cold. in england, in the winter of - , hmpv was detected by rt-pcr in association with . % of samples taken from patients in all age groups with influenza-like illnesses negative for hrsv and influenza viruses. hmpv has been increasingly recognized as cause of acute wheezing in children. one study conducted in finland found hmpv in % of wheezing children, who presented significantly higher levels of il- in nasal secretions as compared to children with hrsv-associated wheezing. a study conducted in brazil found that % of the children with hmpv had wheezing and % had chest indrawing. previous history of asthma has been more frequently associated with hmpv than with hrsv infection and hmpv-infected patients are more often treated with bronchodilators and corticosteroids than hrvs-infected patients. little is known about specific mechanisms of pathogenesis and host immune response in hmpv infections. hmpv is a pathogen of both the upper and lower respiratory tracts. hmpv replicates efficiently in the respiratory tract of monkeys, with virus shedding peaking between days and following infection. serologic data indicates that hmpv infects young individuals, and by the age of virtually all children have become seropositive for the agent; reinfections at later ages are common. , , interestingly, coinfection with hmpv has been reported to correlate with increased severity of hrsv infections. a study conducted in the united kingdom found that this coinfection caused a tenfold increase in the relative risk of admission to the icu for mechanical ventilation in children under with hrsv bronchiolitis. a similar finding was also reported in germany. hmpv can be isolated in llc-mk cells from nasal aspirates or nasopharyngeal swabs. the cytopathic effect, characteristically negative on hemadsorption testing, develops usually late after inoculation (up to days). sensitive rt-pcr assays for this agent have been developed in many different laboratories and have rapidly become standard for hmpv diagnosis. , a real-time pcr assay for hmpv showed to be more sensitive than conventional rt-pcr, even when hybridization was used to increase sensitivity of the detection of amplicons generated by the conventional method. using real-time pcr, hmpv was detected in % of samples collected from patients with ari in australia from march to october that were negative for other pathogens. other than supportive measures, oxygen therapy, bronchodilators, corticosteroids and mechanical ventilation, there is no specific antiviral treatment for this agent. ribavirin is inhibitory for hmpv in vitro. although a hmpv vaccine is not available at this time, the demonstration that hamsters, ferrets, and african green monkeys are susceptible to infection by hmpv, and that hamsters vaccinated with serotype a the epidemiology of acute respiratory tract infection in young children: comparison of findings from several developing countries report of a workshop on respiratory viral infections: epidemiology, diagnosis, treatment and prevention acute respiratory viral infections in ambulatory children of urban northeast brazil longitudinal studies of infectious diseases and physical growth of infants in huascar, an underprivileged peri-urban community in at the edge of development: health crises in a transitional society epidemiology of acute respiratory infections in children of developing countries pan american health organization: acute respiratory infections in the americas the magnitude of mortality from acute respiratory infections in children under years in developing countries acute lower respiratory tract infections in hospitalized patients with diarrhea in dhaka day-care center attendance and hospitalization for lower respiratory tract illness viral respiratory infections in young children attending day care in urban northeast brazil epidemiology and seasonality of respiratory tract virus infections in the tropics the cultural context of breastfeeding: perspectives on the recent decline in breast-feeding in northeast and northcentral brazil reduced mortality among children in southern india receiving a small weekly dose of vitamin a search for a solution: blending oral rehydration therapy (ort) and popular medicine pathogenesis of respiratory infections due to influenza virus: implications for developing countries respiratory viruses predisposing to bacterial infections: role of neuraminidase influenza: emergence and control orthomyxoviridae: the viruses and their replication influenza virus viral vaccines for the prevention of childhood pneumonia in developing nations: priorities and prospects the effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children etiology of acute respiratory infections in children in tropical southern india a community-based study of acute respiratory tract infection in thai children etiology of acute lower respiratory tract infection in children from alabang, metro manilla outbreak of influenza type a (h n ) in iporanga antigenic and genomic relation between human influenza viruses that circulated in argentina in the period - and the corresponding vaccine components regional perspectives on influenza surveillance in africa h n influenza: a protean pandemic threat influenza type a and b infections in hospitalized pediatric patients influenza viruses, cell enzymes, and pathogenicity detection of influenza virus by centrifugal inoculation of mdck cells and staining with monoclonal antibodies rapid detection and simultaneous subtype differentiation of influenza a viruses by real time pcr rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and rational design of potent sialidase-based inhibitors of influenza virus replication efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections resistant influenza a viruses in children treated with oseltamivir: descriptive study centers for disease control and prevention: prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza vaccination in : recommendations and vaccine use in developed and rapidly developing countries immunization against influenza in the elderly: the argentinian experience regional perspectives on influenza surveillance in south america clinical and epidemiologic importance of influenza a viruses resistant to amantadine and rimantadine pandemic influenza: is an antiviral response realistic? respiratory syncytial virus and parainfluenza viruses respiratory syncytial virus brief report: respiratory syncytial virus activity-united states seasonal trends of viral respiratory tract infections in the tropics occurrence and severity of infections caused by subgroup a and b respiratory syncytial virus in children in southeast brazil viral etiology of acute respiratory infections among children in porto alegre, rs, brazil respiratory syncytial virus: changes in prevalence of subgroups a and b among argentinian children clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in sao paulo antigenic characterization of respiratory syncytial virus group a and b isolates in rio de janeiro, brazil management of acute bronchiolitis detection of genomic sequences of respiratory syncytial virus in otitis media with effusion in children acute otitis media in children with bronchiolitis prevalence of serious bacterial infections in febrile infants with respiratory syncytial virus infection etiology of acute lower respiratory tract infection in children from alabang, metro manilla rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization microbial inciters of acute asthma in urban nigerian children winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease pathogenesis of respiratory syncytial virus bronchiolitis-related wheezing pathogenesis of respiratory syncytial virus vaccine-augmented pathology evaluation of quantitative and typespecific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children american academy of pediatrics committee on infectious diseases: reassessment of the indications for ribavirin therapy in respiratory virus infections combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients role of antibody and use of respiratory syncytial virus (rsv) immune globulin to prevent severe rsv disease in high-risk children parainfluenza viruses parainfluenza viral infections in pediatric outpatients: seasonal patterns and clinical characteristics biology of parainfluenza viruses viral croup: a current perspective parainfluenza and influenza virus infections in pediatric organ transplant recipients simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays efficacy of novel hemagglutinin-neuraminidase inhibitors bcx and bcx against human parainfluenza viruses in vitro and in vivo evaluation of combined live, attenuated respiratory syncytial virus and parainfluenza virus vaccines in infants and young children clinical virology comparative susceptibilities of human embryonic fibroblasts and hela cells for isolation of human rhinoviruses rhinovirus antibodies in an isolated amazon indian tribe frequency and natural history of rhinovirus infections in adults during autumn acute respiratory symptoms in adults in general practice computed tomographic study of the common cold detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization peripheral blood mononuclear cells interleukin- and interferon-g production, cytotoxicity, and antigen-stimulated blastogenesis during experimental rhinovirus infection human rhinovirus infection induces airway epithelial cell production of human beta-defensin both in vitro and in vivo rhinovirus detection: comparison of real-time and conventional pcr clinical studies of antiviral agents for picornaviral infections phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray -percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers efficacy of organic acids in hand cleansers for prevention of rhinovirus infections prevention of natural colds by contact prophylaxis with intranasal alpha -interferon fields' virology molecular epidemiology of adenovirus acute lower respiratory infections of children in the south cone of south america ( - ) latent viral infections in airway epithelium diagnostic procedures for viral, rickettsial and chlamydial infections viral infections of humans prevention: outbreak of pharyngoconjunctival fever at a summer camp-north carolina acute otitis media and respiratory virus infection pneumonias in childhood: etiology and response to antimicrobial therapy adenovirus infections in human immunodeficiency virus-positive patients: clinical features and molecular epidemiology etiologic, clinical and pathologic analysis of fatal cases of acute respiratory tract infections in argentinian children under years of age latent adenoviral infection in the pathogenesis of chronic airway obstruction evaluation of a bedside immunochromatographic test for detection of adenovirus in respiratory samples, by comparison to virus isolation, pcr and real-time pcr rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool intravenous ribavirin therapy for disseminated adenovirus infection coronaviridae: the viruses and their replication identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children clinical virology viral infection of humans an outbreak of coronavirus oc respiratory infection in normandy, france seroepidemiologic study of coronavirus infection in brazilian children and civilian adults rhinovirus and coronavirus infection-associated hospitalizations among older adults community study of role of viral infections in exacerbations of asthma in - year old children human coronavirus e infects polarized airway epithelia from the apical surface the effects of coronavirus on human nasal ciliated respiratory epithelium fields' virology. philadelphia association between a novel human coronavirus and kawasaki disease frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infections by use of a novel real-time reversetranscriptase polymerase chain reaction intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers severe acute respiratory syndrome: identification of the etiological agent update: outbreak of severe acute respiratory syndrome worldwide a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and commom mutations associated with putative origins of infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study severe acute respiratory syndrome isolation and characterisation of viruses related to sars coronavirus from animals in southern china sars-related virus predating sars outbreak long-term sars coronavirus excretion from patient cohort epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong sars: responding to an unknown virus clinical manifestations, laboratory findings, and treatment outcomes of sars patients angiotensin-converting enzyme is a functional receptor for the sars coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dentritic cell transfer through dc sign viral loads in clinical specimens and sars manifestations reverse genetics with a full-length infectious cdna clone of severe acute respiratory syndrome coronavirus the aetiology, origins, and diagnosis of severe acute respiratory syndrome evaluation of reverse transcription-pcr assays for repid diagnosis of severe respiratory syndrome associated with a novel coronavirus role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical finings interferon alfacon- plus corticosteroids in severe acute respiratory syndrome use of convalescent plasma therapy in sars patients in hong kong sars among critical care nurses acute respiratory tract infections among a birth cohort of children from cali, colombia, who were studied through months of age etiology of acute lower respiratory tract infections in children in a rural community in the gambia viral agents of acute respiratory infections in young children in kuala lampur viral etiology and epidemiology of acute respiratory infections in children in nairobi, kenya diagnoses of acute lower respiratory tract infections in children in rawalpindi and islamabad a study of nonbacterial agents of acute lower respiratory tract infection in thai children patterns of acute respiratory tract infection in children: a longitudinal study in a depressed community in metro manila characterization of human metapneumoviruses isolated from patients in north america analysis of the genome sequence of a human metapneumovirus human metapneumovirus as a cause of community-acquired respiratory illness antigenic and genetic variability of human metapneumoviruses human metapneumovirusassociated lower respiratory tract infections among hospitalized human immunodeficiency virus type (hiv- )-infected and hiv- -uninfected african infants virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory infections in all age groups human metapneumovirus as a causative agent of acute bronchiolitis in infants human metapneumovirus infection in hospital referred south african children respiratory syncytial virus and metapneumovirus in children over two seasons with a high incidence of respiratory infections in brazil human metapneumovirus infections in the canadian population evidence of human metapneumovirus in children in argentina molecular assays for detection of human metapneumovirus human metapneumovirus in lower respiratory tract disease in otherwise healthy infants and children children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus associated with respiratory tract infections in a -year study of nasal swabs from infants in italy metapneumovirus and acute wheezing in children dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis prospective study of human metapneumovirus infection in children less than years of age identification of small animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hmpv) and implications for hmpv design were protected from challenge with either a or b hmpv serotypes opens possibilities for hmpv vaccine design. humanized neutralizing monoclonal antibody to f protein is active in experimentally infected animals. key: cord- -mxt stat authors: saraya, takeshi; kurai, daisuke; ishii, haruyuki; ito, anri; sasaki, yoshiko; niwa, shoichi; kiyota, naoko; tsukagoshi, hiroyuki; kozawa, kunihisa; goto, hajime; takizawa, hajime title: epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: mxt stat viral respiratory infections may be associated with the virus-induced asthma in adults as well as children. particularly, human rhinovirus is strongly suggested a major candidate for the associations of the virus-induced asthma. thus, in this review, we reviewed and focused on the epidemiology, pathophysiology, and treatment of virus-induced asthma with special reference on human rhinovirus. furthermore, we added our preliminary data regarding the clinical and virological findings in the present review. more than different types of viruses, such as human rhinovirus (hrv), human metapneumovirus (hmpv), respiratory syncytial virus (rsv), and human parainfluenza virus (hpiv), are known to cause acute respiratory illness (ari; tsukagoshi et al., ) . we recently reported the issue of "virus-induced exacerbation in asthma and chronic obstructive pulmonary disease" (kurai et al., a) , however, among these causative viruses, hrv is now recognized to have a major impact on asthma pathogenesis (fujitsuka et al., ) . from this perspective, we reviewed the literature regarding the epidemiology of hrv-induced asthma in adults, together with preliminary epidemiological data obtained at our institution. hrv belongs to the genus enterovirus and family picornaviridae (turner and couch, ) . hrv possesses a single strand positive-sense rna (ssrna) genome of approximately . kb. the viral capsid is composed of four viral proteins (vp - ) which are assembled into protomers, resulting in a small icosahedral structure with a diameter of about - nm (turner and couch, ) . genetically, hrv is classified into three species; hrv-a, -b, and -c (simmonds et al., ) . furthermore, these species of hrv have more than genotypes (andries et al., ; arakawa et al., ; kiyota et al., kiyota et al., , . molecular epidemiological studies suggest that the dominant species are hrv-a and -c, while hrv-b is relatively rarely detected (arakawa et al., ; kiyota et al., ) . in particular, the vp and vp proteins have variations in their amino acid sequences, accounting for the large number of viral serotypes (turner and couch, ) . the host receptor for hrv in respiratory epithelial cells is the intracellular adhesion molecule (icam- , cd ) for the major hrv serotypes (hrv-a and -b), or low-density lipoprotein receptor (ldlr) for the other minor hrv serotypes. the receptor for hrv-c is not yet known. it has been suggested that the optimal temperature for replication of hrv is relatively cool ( - • c), which would limit infections to the upper airway; however, large or medium sized airways lower in the respiratory tract are now also considered cool enough for hrv replication, in spite of the higher temperature of the lung parenchyma ( • c; mcfadden et al., ) . therefore, hrv is potentially a causative agent of more severe ari such as bronchiolitis and pneumonia (turner and couch, ; watanabe et al., ; smuts et al., ; arakawa et al., ) , and may be associated with virus-induced asthma linsuwanon et al., ; fujitsuka et al., ; smuts et al., ) . hrv might therefore be involved in various aris and additional respiratory complications (kiyota et al., ) . lieberman et al. ( ) reported that the detection of any virus include hrv, the sensitivity rates for nasopharyngeal swab ( . %) was superior than that of oropharyngeal swab ( . %), respectively. the common cold is the third most common primary diagnosis in office visits (hsiao et al., ) , and this disease is generally selflimiting, usually lasting up to days (fashner et al., ) . among the general population, hrv infection causes common colds at a frequency of - % (makela et al., ; van gageldonk-lafeber et al., ) . tyrrell et al. ( ) reported that intranasal www.frontiersin.org inoculation with either hrv serotypes , , and , coronavirus type e, or rsv in healthy volunteers induced patterns of symptom development which were not substantially different from each other. however, individual signs or symptoms occurred earliest in hrv infections, then in coronavirus, and lastly in rsv, appearing up to days after inoculation, which demonstrated the long incubation periods of rsv in volunteers (tyrrell et al., ) . hrv has been implicated in patients with acute otitis media, exacerbation of chronic obstructive pulmonary disease, common cold, and lower respiratory tract infections in neonates, the elderly and immunocompromised. arruda et al. ( ) researching the frequency and natural history of hrv infections in adults during autumn, demonstrated that the first symptom noticed most often was sore throat ( %) in hrv culture-or pcr-positive patients, and stuffy nose in hrv-negative patients ( %), using nasal wash specimens. respiratory symptoms typically develop after - days after inoculation in studies, and uncomplicated hrv infections usually peak - days after inoculation. the median duration of hrv colds is week, but up to % last more than weeks (gwaltney et al., ; rotbart and hayden, ) . it should be noted that in illness caused by hrv, viral shedding occurs naturally for up to days, but predominantly over a - days period. hrv-a type (hrv- ), a major group virus commonly used for experimental human infection, and hrv-a type (hrv- ), which has been used in animal models of hrv infection, are closely related. grunberg et al. ( ) reported that experimental hrv- infection via nasal inhalation leads to a transient decrease of fev . in patients with asthma, and this decreased lung function was correlated with enhanced cold symptoms and / or airway hyperresponsiveness. contoli et al. ( ) demonstrated that type iii interferon (ifn-λ) production levels in ex vivo cell cultures derived from bronchial epithelial cells (becs) and macrophages obtained from asthmatic patients, were lower than in those derived from healthy controls. furthermore, deficient interferon-λ production was correlated with hrv viral load, severity of clinical symptoms and fev . . message et al. ( ) demonstrated that the severity of intranasally inoculated hrv-induced clinical illness in asthmatic subjects was correlated to virus load and lower airway virus-induced inflammation. on the other hand, demore et al. ( ) reported that no difference in clinical symptoms, and patterns of viral shedding, was noted between subjects with persistent allergic asthma and healthy subjects after experimental infection with hrv. these different results after experimental hrv infection in individual studies in asthmatic patients and healthy subjects might be dependent on the severity of the asthma of those subjects who enrolled in the studies. indeed, in several reports, neither defective ifn induction by hrv, nor increased hrv replication was observed in primary human becs derived from subjects with well controlled asthma (lopez-souza et al., ; bochkov et al., ; sykes et al., ) . a few animal models for rhinovirus infection have been showed because a major group of hrv (i.e., hrv- ) did not bind mouse icam- . only a minor group of hrv (i.e., hrv- b) infected the mouse. in this regard, bartlett et al. ( ) generated a transgenic balb/c mouse expressing a mouse-human icam- chimeric receptor for hrv- infection. this study also showed asthma exacerbation model by intraperitoneally sensitized with ovalbumin with aluminum hydroxide followed by intranasal inoculation of hrv- b or uv-inactivated hrv- b. although data regarding virus respiratory infections (vris) as precipitators of asthma attacks in adults are less clear, nicholson et al. ( ) reported that vris are as commonly linked to exacerbations in adults as they are in children (johnston et al., ; fujitsuka et al., ) . this study showed that viruses were detected in % of clinical exacerbative episodes with a decrease in peak expiratory flow rate (pefr) of ml/minute or more, and the most commonly identified virus was hrv, followed by coronaviruses and parainfluenza viruses (nicholson et al., ) . thus, the virus most commonly detected in asthma exacerbations appears to be hrv. although hrv is well known as the most frequent cause of the common cold, the implications of hrv infection vary according to respiratory diseases. table shows the frequency of hrv infection in various adult respiratory diseases such as exacerbation of asthma (nicholson et al., ; atmar et al., ; tan et al., ) , common cold (makela et al., ; van gageldonk-lafeber et al., ) , exacerbation of copd (seemungal et al., ; rohde et al., ; tan et al., ; beckham et al., ; papi et al., ; hutchinson et al., ; ko et al., ; mcmanus et al., ; kherad et al., ; dimopoulos et al., ; perotin et al., ) , community acquired pneumonia (jennings et al., ; johnstone et al., ; johansson et al., ; lieberman et al., ; fry et al., ; wootton et al., ; luchsinger et al., ; takahashi et al., ; huijskens et al., ) , exacerbation of idiopathic pulmonary fibrosis (wootton et al., ) , and asymptomatic infection (fry et al., ) . the risk of exacerbations of asthma in adults is elevated after children return to school, and around december th (the christmas holiday in westernized countries), and this is likely to be due to social interactions with children at these times. prospective monitoring studies using reverse transcription polymerase chain reaction (rt-pcr) indicate that as many as % of acute asthma exacerbations in children, and about % in adults, were associated with the presence of upper respiratory tract (urt) infections. corne et al. ( ) found that the detection rates of hrv in asthmatic ( . %) and healthy participants ( . %) were similar, but the lrt symptoms were significantly more severe and longer lasting in the asthmatic group than in the healthy group based on one definition of urt and lrt symptoms ( table ; johnston et al., ) . there is no common antigen across all strains of hrvs; therefore, no reliable diagnostic method for hrv infection has been established using hrv antigens or hrv-specific antibody. although viral culture is the conventional method for hrv detection, culture methods are not practical in clinical settings for the detection of hrv, because of its slow growing character and requirement for specific culture conditions. furthermore, the diagnostic capability of molecular amplification techniques frontiers in microbiology | virology exacerbation of asthma - nicholson et al. ( ) , tan et al. ( ) , atmar et al. ( ) common cold - makela et al. ( ) cited and adapted from johnston et al. ( ) . such as nucleic acid sequence-based amplification and rt-pcr is superior to those of culture methods (loens et al., ) . experimental hrv infections have been shown to lead to a longlasting excessive airway narrowing in volunteer subjects with asthma (cheung et al., ; grunberg et al., ) . of note, rhinovirus, unlike influenza and other viruses, causes minimal cytotoxicity (fraenkel et al., ) , and the amount of epithelial damage does not correlate with the severity of the symptoms. hrv infection can cause additive or synergistic effects in exacerbation of asthma via the influx of additional inflammatory cells in the airways with preexisted inflammation, resulting in airway cholinergic hyperresponsiveness (nagarkar et al., ) , as an allergic response. the effects of hrv infection such as enhanced contractility of airway smooth muscle (asm) cell and impaired relaxation to cholinergic or β-adrenaergic agonists are attributed solely to binding of the virus to its host receptor icam- on the asm cell surface. this proasthmatic-like effect was recognized even in the situation of complete inhibition of viral replication in vitro, but not in the setting of pretreatment of asm with neutralizing antibody directed against for icam- (grunstein et al., ) . thus, the hrv attachment to icam- itself can affects the contractility of asm cells in the absence of any cytopathic effects, and chun et al. ( ) reported that a cells infected with hrv in vitro produced a higher value of il- and rantes than those of rsv or adenovirus. in addition, only the combination of hrv with der f (house dust mites antigen) acted synergistically to induce il- production. these findings are the reason why the hrv can be a major pathogen for acute exacerbation of asthma. we present a schema for pathogenesis in hrv associated asthma exacerbations (figure ) , which requires the following steps, ( ) hrv attachment to airway epithelial cells, ( ) an innate immune response which leads to epithelial damage, ( ) infection-related airway remodeling. when rt-pcr is used to either supplement or replace conventional culture techniques, viruses have been found in approximately one half to three quarters of adults experiencing an acute wheezing episode (jackson and johnston, ) , and the majority ( %) of viruses identified were hrvs (nicholson et al., ) . however, the evidence is weak, and mechanisms are poorly understood. initially, hrv-a and -b attach to airway epithelial cells via icam- or ldlr (kennedy et al., ) . the receptor or receptors for the recently identified group hrv-c have yet to be clarified. hrv-infected becs secrete a wide range of cytokines and chemokines such as il- , il- , ccl /rantes (regulated on activation, normal t cell expressed and secreted), cxcl /il- , gm-csf, and cxcl /interferon-inducible protein (ip- ; jackson and johnston, ; proud, ) , which induce neutrophilic, lymphocytic, and eosinophilic inflammation together with airway hyperresponsiveness and airway remodeling (wark et al., ; proud, ) . clearance of viral pathogens begins with interferon secretion, and the underproduction of these factors has been postulated to lead to viral-induced exacerbations. there are three types of interferons, based on the receptors they bind: type i (ifn-α/β), type ii (ifn-γ), www.frontiersin.org and type iii (ifn-λ). hrv infection induced epithelial expression of mrna for both type i and type iii ifns, and it has been suggested that impaired epithelial production of ifn-β and ifnλ in asthmatic subjects may contribute to viral exacerbations of asthma (wark et al., ; contoli et al., ) . contoli et al. ( ) showed significant inverse correlations between ex vivo production of ifn-λ and severity of symptoms, bronchoalveolar lavage viral load and airway inflammation, and a strong positive correlation with reductions in lung function during in vivo infection. genome-wide association studies showed that single nucleotide polymorphisms involve in various diseases. interferon-λ polymorphisms may effect on the incidence of hrv infection (russell et al., ) . message et al. ( ) reported virus load in asthmatic subjects as being related to increased lower airway inflammation, and in turn increased lower airway inflammation being related to increased symptoms, reductions in lung function, and increases in bronchial hyperreactivity. these data suggest a causal role for hrv infection in the pathogenesis of asthma exacerbations. investigating virus-allergen interactions, durrani et al. ( ) demonstrated that another mechanism that increased expression and cross-linking of the high-affinity ige receptor, fcεri, on plasmacytoid dendritic cells is associated with reduced hrv-induced ifn-α and ifn-λ secretion, and allergic asthmatic children have significantly reduced hrv-induced ifn-α and ifn-λ production after cross-linking of fcεri. type , or inducible, nitric oxide synthase (inos) is the major nos isoform found in epithelial cells and can generate substantial amounts of nitric oxide (no). the no molecules both inhibit the replication of hrv in airway epithelial cells, and suppresses hrv-induced cytokine production (proud, ) . although the measurement of fractional no concentration in exhaled breath (feno) may be used to support the diagnosis of asthma (dweik et al., ) , however, increasing of feno seems to be not always correlated with viral load during the period of hrv infection (sanders et al., ) . other factors such as allergy, allergen exposure, tobacco smoke, particulates, ozone, stress, and infections such as sinusitis commonly contribute to exacerbations of asthma. grainge et al. ( ) reported that repeated bronchoconstriction in asthma promotes airway remodeling, and there is now clear evidence that airway remodeling begins in early childhood, and can be present even before clinical diagnosis of asthma is established (pohunek et al., ) . increasing evidence regarding hrv-induced wheezing or exacerbation of asthma raises the possibility that hrv infections could contribute to the initiation and subsequent progression of airway remodeling, which involves multiple factors such as increased epithelial release of mucin ac (mu ac), activin a, amphiregulin, matrix metalloproteinase (mmp ), epidermal growth factor (egf), fibroblast growth factor (fgf), and vascular endothelial growth factor (vegf). hrv infection upregulates production of muc ac from epithelial cells, which leads to airflow obstruction in asthma (hewson et al., ) . activin a is a member of the tgf-β superfamily and amphiregulin, a member of the egf family, alters repair processes (leigh et al., ) . both activin a and amphiregulin have been linked to subepithelial basement membrane thickening in asthma. mmp appears to have important roles in asthma exacerbation and airway remodeling (sampsonas et al., ) . expression of vegf and its receptors is increased in asthmatic subjects, and vegf is the major proangiogenic activator in asthmatic airways (feltis et al., ; simcock et al., ) . kuga et al. ( ) reported that . % of adult asthmatic patients with common cold suffered an asthma attack, and common cold was significantly associated with acute exacerbations of asthma. they also stated that hrv infection might be important as the virus was detected by rt-pcr in throat gargles (kuga et al., ) . virus-induced exacerbation of asthma is a critical issue for the general physician. however, among asthmatic patients with exacerbative status, distinguishing between those patients which have vris, and those who do not, is difficult. furthermore, epidemiological data regarding adult asthma exacerbations have been sparsely reported. to investigate the prevalence of vri in exacerbations of adult asthma in both hospitalized or not-hospitalized patients, characterization of clinical and radiological findings was performed. a prospective observational cohort study was conducted at kyorin university hospital, tokyo, japan from august to august (kurai et al., b) . all patients with respiratory symptoms associated with exacerbation of asthma were included, and samples were collected by nasopharyngeal or oropharyngeal swab, and subjected to a pcr method to detect common respiratory viruses. the patients who were enrolled consisted of hospitalized (n = ) or not-hospitalized patients (n = ; table ). in these two groups, the subject's backgrounds were similar for age, sex, smoking rates, and duration of illness, however, the measured value of spo was significantly lower in hospitalized patients ( ± . %) than in non-hospitalized patients ( . ± . %). the incidence of vri was significantly higher in the former group ( . %, n = ) than in the latter group ( . %, n = ; p = . ). in the latter group, influenza virus alone was detected in both patients. furthermore, all hospitalized patients ( %, n = ) had wheezing or severe exacerbation based on the ats (american thoracic society)/ers (european respiratory society) statement (reddel et al., ) , whereas, among nonhospitalized patients, only nine patients ( %) were considered as having a severe exacerbation (p < . ), and patients ( . %) had wheezing (p < . ). these findings suggested that virus infection was certainly associated with the hypoxemia and / or wheezing which resulted in a severe or serious asthma attack, based on the japanese guidelines (ohta et al., ) or the ats/ers statement (reddel et al., ) . previous studies using ohta et al. ( ) , † † defined by reddel et al. ( ) . ** p < . , *** p < . . all data are presented as (mean ± sd). pcr-based viral diagnostics found that viral respiratory infections were detected in up to % of exacerbations of asthma in children and about % of exacerbations in adults (nicholson et al., ; johnston et al., ) , which is similar to our results. serum inflammatory or allergic markers are not different between the hospitalized and non-hospitalized patients ( table ) . in hospitalized patients, the viruses identified were hrv (n = ), hmpv (n = ), and rsv (n = ). at the time of admission, the virus-positive group (n = ) had significant lower values of spo ( . ± . %) than those of the virus-negative group (n = , spo : . ± . %, p < . ), and for the patients whose data are available, the frequency of hypercapnea (paco torr) was significantly higher in the virus positive group ( . %, n = ) than in the virus negative group ( %; p = . ; table ). the mechanisms for hypercapnea in virus infected individuals have not been elucidated. however, cheung et al. ( ) reported that hrv infection causes long lasting excessive airway narrowing in response to methacholine in asthmatic subjects. we speculated that smooth muscle might have a role in exaggerated airway narrowing in virus positive asthmatic patients, as described by king et al. ( ) . interestingly, the incidence of ground glass opacities (ggo) on high resolution computed tomography seemed to be higher for virus-positive hospitalized patients than for virus-negative patients, but it did not reach statistical significance. for example, figure a shows a patchy ggo with thickening of interlobular septa in a -year-old woman who was admitted during an asthma attack induced by hrv-a. figure b also shows ggo in a -year-old man with an asthma attack caused by hrv-c infection. these ggo in both patients could only be detected in hrct, not in chest x-ray. these results suggested that hrv was the major cause of virusinduced asthma, and was possibly involved in lower airway or lung parenchyma features, appearing as ggo. viral infection significantly exaggerated the respiratory status (low spo and hypercapnea) when compared to that of virus-negative asthma exacerbative patients at the time of admission. indeed, in recent years, hrv has been recognized as a common cause of hospital admission, both as an agent of bronchopneumonia and through exacerbation of chronic pulmonary conditions, even in the elderly over years of age (pierangeli et al., ) . curiously, after initiation of treatment with intravenous steroid, both the virus-positive and -negative groups had no significant difference in duration of respiratory failure, wheezing, days in hospital, and even in the time required for steroid treatment. no established treatment for prevention of hrv-induced asthma is available, and we describe the exploratory interventions as follows. inhaled corticosteroid (ics) is the main drug for regular asthma therapy. ics treatment improved airway hyperresponsiveness in asthmatic patients experimentally challenged with hrv, however, ics treatment did not reduce accumulation of inflammatory cells, except for eosinophils in bronchial epithelium (grunberg et al., ) . double-stranded rna (dsrna), a viral product and a ligand for the toll-like receptor- (tlr ), upregulates the expression of inflammatory chemokines in airway epithelial cells. matsukura et al. ( ) reported that treatment of beas- b cells with fluticasone propionate significantly and dose-dependently inhibited dsrna-induced expression of ccl , cxcl , and cxcl protein and mrna. to confirm the effect on ssrna, such as that of hrv, would need further studies. leukotriene receptor antagonist was prescribed in asthmatic patients with or without ics. montelukast treatment did not improve asthma control or cold symptom scores when hrv were experimentally inoculated into mild asthmatics, or healthy subjects (kloepfer et al., ) . it is uncertain whether leukotriene receptor antagonist treatment is effective in the reduction of asthma symptoms associated with hrv infection. zambrano et al. ( ) reported that high serum ige levels in mildly asthmatic children with experimental hrv infection may be associated with enhanced lower respiratory symptoms and elevation of inflammatory markers, such as nasal eosinophil cationic protein and expired nitric oxide, than those of healthy subjects and/or low ige asthmatic patients. the prevalence of asthma was closely associated with the serum ige levels standardized for age and sex (burrows et al., ) , and airway hyperresponsiveness appears to be closely linked to the allergic diathesis, as reflected by the serum total ige level (sears et al., ) . omalizumab, an anti-ige monoclonal antibody, was indicated in inadequately controlled moderate-to-severe persistent allergic asthma patients who were treated with high dose ics. durrani et al. ( ) showed that the ige receptor fcεri is inversely associated with ifn-α and ifn-λ secretion when plasmacytoid dendritic cells derived from allergic asthmatic children were challenged with hrv. omalizumab downregulates fcεri expression on dendritic cells (prussin et al., ) , which may reduce exacerbation of asthma associated with increased production of ifns, through fcεri. no drugs are clinically used in hrv infection, although several drugs have been tried for treatment and prevention of hrv infection. these drugs are summarized in a review (jacobs et al., ) . ifns had a potential protective role in viral induced asthma (cakebread et al., ; gaajetaan et al., ) . becker et al. ( ) showed that exogenous ifn-α, ifn-β, ifn-λ , and ifn-λ inhibited hrv replication in becs from healthy donors. macrolides are known to possess anti-inflammatory and immunomodulatory actions extending beyond their antibacterial activity in pulmonary inflammatory disorders (takizawa et al., ; min and jang, ) . erythromycin inhibits hrv infection by reducing icam- expression on the surface of human tracheal epithelial cells, and modulates inflammation by suppressing the production of proinflammatory cytokines (suzuki et al., ) . yamaya et al. ( ) reported that the mucolytic drug ambroxol hydrochloride, antibiotic drug of levofloxacin (yamaya et al., b) , and bronchodilators (tiotropium, tulobuterol, and procaterol) for asthma or copd (yamaya et al., (yamaya et al., , a (yamaya et al., , may have a beneficial effect in hrv infection, by inhibiting hrv replication and partly reducing icam- expression and acidic endosome production, via the inhibition of nf-kappab activation (yamaya, ) . we reviewed the previous reports regarding hrv-induced asthma exacerbations, together with our results from an institutional prospective study. hrv is a major pathogen for asthma exacerbations, and certainly associated with more serious clinical conditions such as hypoxemia or hypercapnea in hospitalized patients. further accumulation of evidence of virus-induced asthma for multidisciplinary assessment would be helpful for physicians in recognizing the condition or understanding the pathogenic mechanisms. two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity molecular epidemiological study of human rhinovirus species a, b and c from patients with acute respiratory illnesses in japan frequency and natural history of rhinovirus infections in adults during autumn respiratory tract viral infections in inner-city asthmatic adults mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation effect of exogenous interferons on rhinovirus replication and airway inflammatory responses respiratory viral infections in patients with chronic, obstructive pulmonary disease rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma association of asthma with serum ige levels and skin-test reactivity to allergens exogenous ifn-beta has antiviral and anti-inflammatory properties in primary bronchial epithelial cells from asthmatic subjects exposed to rhinovirus rhinovirus inhalation causes long-lasting excessive airway narrowing in response to methacholine in asthmatic subjects in vivo rhinovirus-infected epithelial cells produce more il- and rantes compared with other respiratory viruses role of deficient type iii interferon-lambda production in asthma exacerbations frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study similar colds in subjects with allergic asthma and nonatopic subjects after inoculation with rhinovirus- viral epidemiology of acute exacerbations of chronic obstructive pulmonary disease innate immune responses to rhinovirus are reduced by the highaffinity ige receptor in allergic asthmatic children an official ats clinical practice guideline: interpretation of exhaled nitric oxide levels (feno) for clinical applications treatment of the common cold in children and adults increased vascular endothelial growth factor and receptors: relationship to angiogenesis in asthma lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness interferon-beta induces a long-lasting antiviral state in human respiratory epithelial cells effect of bronchoconstriction on airway remodeling in asthma rhinovirus-induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during experimental infection experimental rhinovirus infection causes variable airway obstruction in subjects with atopic asthma rhinovirus elicits proasthmatic changes in airway responsiveness independently of viral infection rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response rhinovirus induces muc ac in a human infection model and in vitro via nf-kappab and egfr pathways national ambulatory medical care survey: summary the value of signs and symptoms in differentiating between bacterial, viral and mixed aetiology in patients with communityacquired pneumonia a community-based, time-matched, case-control study of respiratory viruses and exacerbations of copd the role of viruses in acute exacerbations of asthma human rhinoviruses incidence and characteristics of viral community-acquired pneumonia in adults etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis community study of role of viral infections in exacerbations of asthma in - year old children pathogenesis of rhinovirus infection upper-respiratory viral infection, biomarkers, and copd exacerbations the mechanics of exaggerated airway narrowing in asthma: the role of smooth muscle genetic analysis of human rhinovirus species a to c detected in patients with acute respiratory infection in kumamoto prefecture genetic analysis of the vp /vp coding region in human rhinovirus species c in patients with acute respiratory infection in japan effects of montelukast on patients with asthma after experimental inoculation with human rhinovirus viral etiology of acute exacerbations of copd in hong kong the correlation between the exacerbation of bronchial asthma and picornavirus (human rhino virus) infection in throat gargles by rt-pcr virusinduced exacerbations in asthma and copd respiratory viral infection in admitted adult patients human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling respiratory viruses in adults with community-acquired pneumonia high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season in vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects community-acquired pneumonia in chile: the clinical relevance in the detection of viruses and atypical bacteria viruses and bacteria in the etiology of the common cold basic research on virus-induced asthma exacerbation: inhibition of inflammatory chemokine expression by fluticasone propionate thermal mapping of the airways in humans respiratory viral infection in exacerbations of copd rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production macrolide therapy in respiratory viral infections rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages respiratory viruses and exacerbations of asthma in adults japanese guideline for adult asthma infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations detection of multiple viral and bacterial infections in acute exacerbation of chronic obstructive pulmonary disease: a pilot prospective study rhinovirus frequently detected in elderly adults attending an emergency department markers of eosinophilic inflammation and tissue re-modelling in children before clinically diagnosed bronchial asthma nitric oxide and the common cold role of rhinovirus infections in asthma. asian pac omalizumab treatment downregulates dendritic cell fcepsilonri expression statement: asthma control and exacerbations: standardizing endpoints for clinical asthma trials and clinical practice respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study picornavirus infections: a primer for the practitioner interferon lambda genetic polymorphisms and viral infection: the tip of the iceberg? dna sequence variations of metalloproteinases: their role in asthma and copd role of nasal nitric oxide in the resolution of experimental rhinovirus infection relation between airway responsiveness and serum ige in children with asthma and in apparently normal children respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease proangiogenic activity in bronchoalveolar lavage fluid from patients with asthma proposals for the classification of human rhinovirus species c into genotypically assigned types human rhinovirus infection in young african children with acute wheezing erythromycin inhibits rhinovirus infection in cultured human tracheal epithelial cells rhinovirus-induced interferon production is not deficient in well controlled asthma the incidence and aetiology of hospitalised community-acquired pneumonia among vietnamese adults: a prospective surveillance in central vietnam erythromycin suppresses interleukin expression by human bronchial epithelial cells: a potential mechanism of its anti-inflammatory action epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease molecular epidemiology of respiratory viruses in virus-induced asthma rhinovirus signs and symptoms in common colds a case-control study of acute respiratory tract infection in general practice patients in the netherlands asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma rhinovirus species and their clinical presentation among different risk groups of non-hospitalized patients viral infection in acute exacerbation of idiopathic pulmonary fibrosis virus infection-induced bronchial asthma exacerbation inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofloxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells procaterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells tulobuterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to ige the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -ft lkpzq authors: proud, david title: upper airway viral infections date: - - journal: pulm pharmacol ther doi: . /j.pupt. . . sha: doc_id: cord_uid: ft lkpzq upper airway viral infections (uri) are a major cause of absence from school and work. although morbidity is low in most of the subjects, the complications of uri, including otitis media, sinusitis and exacerbations of asthma and chronic obstructive pulmonary disease (copd) have an enormous health impact. despite the major health care consequences associated with these complications, our understanding of how uri trigger upper airway symptoms and cause exacerbations of lower airway diseases remains limited. this article reviews our current understanding of the pathogenesis of uri, and of viral exacerbations of asthma and copd, and considers host defense parameters that may regulate susceptibility to disease exacerbations. we will also consider current and potential therapeutic approaches for the treatment of uri and their lower airway complications. their complications uri, manifesting as the clinical syndrome we refer to as the common cold, is the most frequent acute respiratory illness experienced by humans. adults will experience to colds each year, while children experience to . as a result of this, according to the centers for disease control and prevention, million school days are lost annually in the united states due to colds. although common colds can be caused by a variety of different virus types, including coronaviruses, parainfluenza virus and respiratory syncytial virus (rsv), the predominant viral pathogens, particularly in the autumn season, are human rhinoviruses (hrv) [ ] [ ] [ ] . although simple colds in healthy individuals are associated with little morbidity, it has long been known that rhinovirus infections can precipitate or exacerbate other diseases, including otitis media [ ] , and sinusitis [ , ] . more recently, growing evidence also has implicated uri as the predominant risk factor associated with exacerbations of both asthma and chronic obstructive pulmonary disease (copd). in the case of asthma, there is a clear temporal relationship between increase in hospitalizations for asthma exacerbations and outbreaks of uri [ , ] , with a major spike in early september, which is the peak time for hrv infections. moreover, prospective studies using rt-pcr to assist in viral detection have demonstrated that common respiratory viruses are associated with up to % of asthma exacerbations in adults and over % of exacerbations in children [ , ] . although several viral types were found during these exacerbations, the dominant pathogen detected was hrv. hrv also was the most common viral pathogen associated with asthma attacks in young children over years of age presenting in the emergency room [ , ] . there also has been a growing appreciation regarding the important role of uri in triggering exacerbations of copd [ ] . recent studies indicate that about half of all copd exacerbations are associated with viral infections, and that hrv is, again, the dominant viral pathogen [ , ] . interestingly, respiratory viral infections are associated with copd exacerbations that are more frequent, severe and have longer recovery times [ ] . the ability of uri to serve as precipitants for exacerbations of asthma and copd has enormous consequences in terms of both health care costs and patient's quality of life. the total health care costs for asthma in the united states for the year was estimated at $ . billion [ ] , and acute exacerbations account for half of the total health care costs for the disease [ , ] . similarly, acute exacerbations of copd are a major cause of hospitalizations and death, and account for % of health care costs for the disease [ ] . moreover, exacerbation frequency is a major determinant of health status and quality of life for copd patients [ ] . despite the high incidence and serious complications of uri, the mechanisms by which viruses induce upper airway symptoms, or cause exacerbations of lower airway diseases, remain poorly understood. although it is possible that different viral types may induce these outcomes via variable mechanisms, it seems more likely that common aspects of viral pathogenesis dominate. given that hrv is the major viral pathogen associated with colds and exacerbations of asthma and copd, we will focus on the current status of our knowledge of the response to hrv infection as representative of viral pathogenesis, indicating differences with other viral types when appropriate. it is clear that uri are associated with increased airway inflammation. in particular, hrv infections lead to increased numbers of neutrophils and lymphocytes in the upper airways [ ] [ ] [ ] . hrv infections also induce neutrophilic recruitment to the lower airways in subjects with asthma [ , ] . consistent with this virally induced pattern, many acute asthma exacerbations seen in the clinical setting are characterized by increased levels of neutrophils and lymphocytes in the airways [ ] [ ] [ ] . asthmatics who display this neutrophilic profile show a poor response to inhaled corticosteroids [ ] . similarly, while stable copd is associated with a characteristic infiltration of the bronchial mucosa with cd + t lymphocytes and macrophages, severe exacerbations of copd are associated with increased neutrophilic and lymphocytic influx [ , ] . it seems reasonable, therefore, to infer that viruses may trigger exacerbations of asthma and copd by enhancing already existing inflammation in the lower airways. the mechanisms by which viral infections are able to enhance upper, and lower, airway inflammation are not fully defined, but growing evidence supports the concept that viral modulation of epithelial function may initiate the inflammatory response. the airway epithelial cell is the primary target for inhaled pathogens and expresses receptors for several viral types. indeed, the epithelial cell is the only cell type in which hrv has been detected, thus far, by in situ hybridization [ , ] , during in vivo infections. moreover, there is now strong evidence that, upon experimental nasal inoculation with hrv, virus spreads to infect epithelial cells in the lower airways [ , ] , suggesting that epithelial infection may also directly initiate lower airway inflammatory responses. in contrast to viruses such as influenza and rsv, hrv infections do not cause overt epithelial toxicity [ , ] . thus, while the cytotoxic effects of influenza and rsv may contribute to the severity of symptoms, it seems reasonable to assume that alterations of epithelial biology represent a common pathway of symptom development by multiple virus types. in support of this concept, infection of epithelial cells by hrv has been shown to generate a wide variety of proinflammatory chemokines and cytokines, including il- (cxcl ), ena- (cxcl ), ip- (cxcl ), rantes (ccl ), il- , il- and il- [ , [ ] [ ] [ ] [ ] [ ] [ ] . given that several of these products also are detected in airway secretions during hrv infections in vivo [ , , [ ] [ ] [ ] , it is likely that they contribute to recruitment and activation of inflammatory cells during infections. the ability to induce proinflammatory cytokine production from epithelial cells is also shared by other viruses. for example, influenza infection induces epithelial production of il- , il- and rantes [ ] , while rsv infections induce expression of a wide range of chemokine genes [ ] . although there is a clear potential for these chemokines and cytokines to induce inflammation, the profile of products described clearly has the capacity to recruit a plethora of inflammatory cell types to the airways. despite this, a relatively selective cellular profile is seen during infections in vivo. it is unclear what other parameters regulate this limited pattern of inflammatory cell recruitment. it also must be acknowledged that our understanding of the mechanisms by which virally induced chemokine production occurs remains limited. in the case of hrv infections, some chemokines are induced quickly after viral exposure and do not seem to require viral replication [ , ] , while other genes are not induced until several hours post-infection and are absolutely dependent upon replicating virus [ , ] . although both article in press phosphatidylinositol -kinase and mitogen activated protein kinase pathways have been implicated in viral induction of chemokines [ ] [ ] [ ] , the early signaling events induced by virus remain poorly elucidated. similarly, while the viral replication intermediate, double-stranded rna, and the rhinovirus c protease both have been implicated as mediators of late, replication dependent events [ , , ] , the pathways by which such products induce responses are not well defined. moreover, while it is clear that viral induction of some cytokines and chemokines occurs via transcriptional pathways involving nf-kb and/ or interferon regulatory factor [ , ] , our understanding of the control of transcriptional and post-transcriptional regulation of epithelial cytokine and chemokine production in response to viral infection also is limited and requires further study. delineating those aspects of signaling that may be unique for viral induction of chemokines may provide a rational basis for targeted interventions, while studies with selective chemokine or chemokine receptor antagonists will be required to provide a definitive answer on which are the key epithelial mediators involved in disease pathogenesis. once viral infection of the epithelium initiates a proinflammatory process, subsequent production of other mediators that are not of epithelial origin may further contribute to the inflammatory status of the airways. these mediators may be derived from plasma or from other cell types within the airway mucosa. of the mediators assessed thus far, some are relatively virus-specific, while others are observed with colds induced by multiple viral types. for example, while rsv infections have been reported to be associated with the generation of virus-specific ige and increased release of histamine into nasal secretions [ ] , levels of histamine are not increased in airway secretions during hrv infections [ ] . moreover, while older antihistamines that display anticholinergic and sedative properties do reduce rhinorrhea during colds, second generation antihistamines lacking these side effects are ineffective [ ] . by contrast, other mediators, such as kinins and leukotrienes have been associated with infections due to more than one type of common respiratory virus [ , [ ] [ ] [ ] . defining the role of each of these mediators in disease pathogenesis will, again, require studies with effective and selective antagonists. several factors are likely to play a role in determining the severity of the clinical outcome to upper airway viral responses, including the susceptibility of patients with asthma or copd to experience lower airway exacerbations. such factors include pre-existing immunity to a particular viral strain and, in the case of lower airways, the degree of disease control at the time of infection. another important factor is likely to be the variability of the individual host immune and antiviral response to infection. although both innate and specific immunity contribute to the host response to infection, it appears as though the innate response is dominant early after infection and is more likely to help regulate the symptomatic response. for example, while hrv infections elicit antigen-specific humoral and cellular immune responses, these are usually not detectable until after disease symptoms have resolved [ ] . as may be expected based on its central role in viral infection, the epithelial cell is a significant contributor to the innate response to infection. as noted above, infected cells release several cytokines and chemokines that can recruit, and activate, cells of the immune system to the airways. in addition, epithelial production of nitric oxide (no) appears to play an important role in host antiviral responses [ ] . infection of cultured epithelial cells with any of several common respiratory viruses leads to marked upregulation of inducible nitric oxide synthase (inos) and increased generation of no. a similar response occurs during experimental hrv infections in vivo, in that levels of epithelial inos induction correlate with levels of exhaled no. interestingly, in this study, subjects with the highest levels of exhaled no cleared virus more rapidly and had lower symptoms [ ] . a rationale for this is provided by several studies showing that no exerts direct antiviral activity against several common respiratory viruses, in part by nitroslyating key thiol residues in viral proteases. moreover, it has been shown that no also inhibits virally induced generation of several cytokines and chemokines from epithelial cells [ ] . it also should be noted that infection of epithelial cells with hrv induces the production of human b-defensin- (hbd- ) and hbd- [ ] . these peptides are chemotactic for immature dendritic cells expressing ccr , as well as other cell types contributing to the immune response, and likely play an important role in linking innate and specific immunity to hrv [ ] . hbd- also can reduce the extent of influenza infections by blocking the fusion of the virus with cell membranes [ ] . as viral replication progresses, and intact virus is released into airway secretions, it can interact with other cell types that may further contribute to the immune response. presumably, for example, dendritic cells initiate antigen presentation to t cells in the airways or lymph nodes to initiate the specific immune response. in addition, monocytes and macrophages may release additional cytokines, including interferons that can stimulate a variety of interferon (ifn)-stimulated genes (isgs) that collectively limit virus replication and spread. traditional approaches to mitigate the effects of uri have focused on symptomatic relief, although such approaches have generally had modest success. the topical anticholinergic, ipratropium bromide reduces rhinorrhea, an effect mimicked to some degree, as noted above, by older ''first generation'' antihistamines that are known to also have anticholinergic properties. similarly oral adrenergic drugs have modest benefit in terms of reducing nasal blockage, while topical agents have a greater efficacy but suffer from issues of rebound [ ] . a somewhat greater reduction in total symptoms was observed in experimental hrv infections when a combination of ifn-a b, together with the first generation antihistamine, chlorpheniramine, and ibuprofen was administered beginning h after viral challenge [ ] . although this represents an important proof of concept regarding the effectiveness of combining an antiviral compound with conventional compounds for symptomatic relief, the practical utility of this combination is limited by cost factors. there is also a growing literature on the use of ''natural' remedies for the treatment of colds. although the rationale for the use of zinc in the treatment of colds is not well established, multiple studies have evaluated the effectiveness of various zinc salts in this regard. overall, the results of these studies have been inconclusive. a recent, wellcontrolled trial, however, found that zinc salts had an extremely modest effect in reducing symptoms of experimental hrv infections and was ineffective in natural colds [ ] . similarly, echinacea and ginseng have been widely reported as herbal remedies for colds. most of the trials to evaluate such agents have been small and have generated conflicting data. recent randomized trials with relatively large number of subjects reported modest efficacy of a ginseng extract in reducing the frequency of natural colds [ ] , but found no significant effect of an extract of echinacea in experimental hrv infections [ ] . a major issue in regard to trials of herbal remedies, of course, is that there is no standardization of extracts used across studies. indeed, given that the identity of any proposed ''active'' ingredient is unknown, it is impossible to know what to standardize. all of the above trials have been limited to analyzing effects on nasal symptoms, and have never evaluated effects on viral exacerbations of asthma or copd. given the data reported, however, it seems unlikely that they will provide any major benefit. indeed, an interesting question is whether drugs that are currently used for the treatment of asthma and copd have any beneficial effects during viral exacerbations. there is no doubt that the use of corticosteroids, long acting b-agonists, or leukotriene receptor antagonists, alone or in combination, to maintain optimal asthma control has proven efficacious in reducing number of asthma exacerbations, and the use of oral corticosteroids early in exacerbations helps prevent relapses. effects on basal inflammation, however, may not necessarily translate to effects on viral-specific inflammation, and there have been no defined studies of the effects of these medications during asthma or copd exacerbations of known viral origin. corticosteroids have little efficacy in hrv-induced colds [ ] , and it is of interest that asthmatics who display prominent sputum neutrophilia, perhaps indicative of viral etiology, are poorly responsive to inhaled corticosteroids [ ] . because of these limitations, alternative therapeutic approaches to virally induced airway disease continue to be sought. an obvious strategy is to use antiviral approaches. influenza vaccine is clearly effective in reducing upper airway symptoms, and in preventing lower disease exacerbations, induced by this virus during the winter months. unfortunately, vaccination approaches are not feasible for hrv, given the large number of viral serotypes, and have, thus far, proven unsuccessful in the case of rsv infections. neutralizing antibody prophylaxis has proven effective in preventing rsv-induced bronchiolitis but cost factors limit a broader utility and, again, major feasibility issues would arise with using this approach for hrv. selective antiviral approaches have shown more promise. in the case of influenza, neuraminidase inhibitors have been available for several years and have proven clinical efficacy in reducing development and severity of symptoms. at least two approaches also have been used to develop potential antiviral agents against hrv. the novel capsid-binding inhibitor, pleconaril, prevents viral uncoating. in natural colds, oral pleconaril ( mg) administered three times daily led to a significant, but modest reduction in symptoms and also shortened the reported duration of colds [ ] . concerns regarding effects on cytochrome p a , however, precluded further development as an oral treatment. the alternative antiviral approach used for hrv infections has targeted inhibition of the viral c protease, which is necessary for cleavage of the viral polyprotein and, thus, replication. topical administration of one c inhibitor, ruprintrivir, significantly inhibited symptoms in experimental hrv infections even when administered beginning h after infection, although multiple daily dosing was required [ ] . neither of these drugs has been evaluated for their ability to limit hrvinduced exacerbations of lower airway diseases. upper respiratory tract viral infections and their complications lead to a significant burden on health care systems throughout the world. current treatments are less than ideal, and a greater insight into the molecular basis by which common viruses induce both upper and lower airway symptoms is needed if alternative therapeutic approaches are to be developed rationally. the delineation of which specific cytokines and chemokines are key contributors to disease pathogenesis, and elucidation of signaling pathways selective for viral modulation of epithelial cell function may identify novel targets for therapy. alternatively, endogenous enhancement of key host antiviral and host defense molecules, or the topical administration of such molecules may provide alternative approaches to reduce the sequelae of viral infection. upper respiratory viral infections (uri) and their complications rhinovirus infections in an industrial population. i. the occurrence of illness the tecumseh study of respiratory illness. ii. pattern of occurrence of infection with respiratory pathogens, - frequency and natural history of rhinovirus infections in adults during autumn rhinovirus in otitis media with effusion physiologic abnormalities of the paranasal sinuses during experimental rhinovirus colds computed tomographic study of the common cold the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis the september epidemic of asthma hospitalization: school children as disease vectors respiratory viruses and exacerbations of asthma in adults community study of role of viral infections in exacerbations of asthma in - year old children risk factors in acute wheezing in infants and children: viruses, passive smoke, and ige antibodies to inhalant allergens rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses exacerbations of chronic obstructive pulmonary disease respiratory viruses, symptoms and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study an economic evaluation of asthma in the united states a national estimate of the economic costs of asthma global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. nhlbi/who global initiative for chronic obstructive lung disease (gold) workshop summary effect of exacerbation of quality of life inpatients with chronic obstructive pulmonary disease nasal secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection kinins are generated during experimental rhinovirus colds human airway epithelial cells produce ip- (cxcl ) in vitro and in vivo upon rhinovirus infection inflammatory indices in induced sputum: a feasibility study the effect of an experimental rhinovirus infection on bronchial lavage neutrophils prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation increased neutrophil numbers and il- levels in airway secretions in acute severe asthma. clinical and biologic significance inflammatory events in severe acute asthma analysis of induced sputum in adults with asthma: identification of subgroup with isolated sputum neutrophilia and poor response to inhaled corticosteroids biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease changes in sputum t-lymphocyte subpopulations at the onset of severe exacerbations of chronic obstructive pulmonary disease detection of rhinovirus infection of the nasal mucosa by oligonucleotide in situ hybridization localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization rhinoviruses infect the lower airways quantitative and qualitative analysis of rhinovirus infection in bronchial tissues light and scanning electron microscopy of nasal biopsy material from patients with naturally acquired common colds infection of a human respiratory epithelial cell line with rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure rhinovirus induction of the cxc chemokine epithelialneutrophil activating peptide- in bronchial epithelium rhinovirus replication causes rantes production in primary bronchial epithelial cells rhinovirus infection of primary cultures of human tracheal epithelium: role of icam- and il- b nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line interleukin- : stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness increased levels of interleukin- are detected in nasal secretions of volunteers during experimental rhinovirus colds macrophage-inhibitory protein a, and the eosinophil product major basic protein are released into upper respiratory secretions during virus-induced asthma exacerbations in children expression of il- , il- , and rantes on human bronchial epithelial cells, nci-h , induced by influenza virus a expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cdna microarrays phosphatidylinositol -kinase is required for rhinovirusinduced airway epithelial cell interleukin- expression role of p mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells mapk activation is involved in posttranscriptional regulation of rsv-induced rantes gene expression rhinovirus c protease induces interleukin- and granulocytemacrophage colony-stimulating factor expression in human bronchial epithelial cells rhinovirus induces airway epithelial gene expression through doublestranded rna and ifn-dependent pathways multiple cis regulatory elements control rantes promoter activity in alveolar epithelial cells infected with respiratory syncytial virus the development of respiratory syncytial virus specific ige and the release of histamine in nasopharyngeal secretions after infection the treatment of rhinovirus infections: progress and potential kinins are generated in nasal secretions during influenza a infections in ferrets the release of leukotrienes in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease elevations of local leukotriene c levels during viral upper respiratory tract infections the time course of the humoral immune response to rhinovirus infection nitric oxide and the common cold role of nasal nitric oxide in the resolution of experimental rhinovirus infection human rhinovirus infection induces airway epithelial cell production of human b-defensin- both in vitro and in vivo the role of defensins in virus-induced asthma carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins combined antiviral-antimediator treatment for the common cold effect of treatment with zinc gluconate or zinc acetate on experimental and natural colds efficacy of an extract of north american ginseng containing poly-furanosylpyranosyl-saccharides for preventing upper respiratorytract infections: a randomized controlled trial an evaluation of echinacea angustifolia in experimental rhinovirus infections a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of double-blind, randomized, placebo-controlled trials phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray -percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers key: cord- -b w zrl authors: nan title: oeld/population health sig: poster session date: - - journal: respirology doi: . /j. - . . _ .x sha: doc_id: cord_uid: b w zrl nan patients with non-eosinophilic asthma (nea) or copd have increased numbers of neutrophils in the airways. we have shown a similar defect in the ability of alveolar macrophages (am) to phagocytose apoptotic cells, in sputum from patients with nea and copd. we have also shown that bal-derived am from patients with copd have reduced expression of key macrophage phagocytic recognition molecules. the aim of this pilot study was to investigate the expression of these macrophage markers in induced sputum from patients with eosinophilic asthma (ea, n = ), nea (n = ), copd (n = ) and controls (n = ). methods participants underwent clinical assessment, skin allergy test, hypertonic saline challenge and sputum induction. macrophage phagocytosis of apoptotic cells, expression of mannose receptor (mr), hspr (cd ) and pcam (cd ) was determined using fl ow cytometry. results phagocytosis was signifi cantly impaired in patients with nea and copd. expression of mr, cd and cd were decreased in patients with nea or copd, but not signifi cantly changed in ea conclusion impaired sputum-macrophage phagocytosis of apoptotic cells in nea is associated with reduced expression of key macrophage recognition molecules. this defect may contribute to the chronic infl ammation and persistent airway neutrophilia that characterizes this asthma subtype. the use of induced sputum as a surrogate for the more-invasive bronchoscopic sampling may provide a tool for investigating the mechanisms for the effect of therapies including azithromycin in lung disease. supported by nhmrc. neutrophilic asthma (na) has been associated with increased bacterial colonization of the airways and increased expression of innate immune factors in the lung. this suggests that infection may play an important role in the pathogenesis of na. na is an important health issue as sufferers are resistant to steroid treatment, which is the mainstay of asthma therapy and effective therapies are urgently required. using mouse models of chlamydia and haemophilus infl uenzae lung infection and ovalbumin (ova)-induced allergic airway disease (aad), we have shown how infection may be linked to na. both infections suppressed eosinophilic infl ammation and t-helper (th) type responses but increase neutrophilic infl ammation and innate and th and/or th responses in aad. in the current study, the effectiveness of steroid treatment for the suppression of infection-induced neutrophilic aad was assessed by treating infected ovasensitized mice intranasally with dexamethasone during ova challenge. whilst dexamethasone treatment suppressed th -mediated, eosinophilic aad in uninfected, ova-sensitized groups, chlamydia and haemophilus-induced neutrophilic aad were shown to be steroid-resistant. our fi ndings correlate with clinical observations which show associations between infection, neutrophilic infl ammation and steroid resistance in asthmatics. these models will be utilized to examine the effectiveness of a number of novel therapies for infection-induced neutrophilic aad and to develop improved treatment strategies for steroid-resistant asthma. supported by nhmrc, asthma foundation of nsw, hmri. kj baines , , jl simp s on , , rj scott , lg wood , , pg gibson , priority research centre's for asthma and respiratory disease, and information based medicine, the university of newcastle, nsw, australia, and respiratory & sleep medicine, hmri, john hunter hospital, nsw, australia rationale four infl ammatory phenotypes of asthma have been identifi ed including eosinophilic, neutrophilic, mixed granulocytic and paucigranulocytic asthma, based on the presence or absence of sputum granulocytes. the involvement of systemic infl ammation in the pathogenesis of infl ammatory phenotypes of asthma remains unknown. objective this study investigates differences in the whole genome gene expression profi le of peripheral blood in infl ammatory phenotypes of asthma. methods induced sputum and peripheral blood were collected from participants with asthma (n = ). infl ammatory cell counts were performed and infl ammatory phenotype assigned based on the eosinophil and neutrophil cutoffs of % and %, respectively. rna was extracted from whole blood, gene expression profi les were generated (illumina humanref- v ) and analysed using genespring gx . results participants with eosinophilic asthma had signifi cantly higher rates of atopy and levels of exhaled nitric oxide. there were genes classifi ed as differentially expressed between the asthma phenotypes including the α-defensins (defa) , b, and , neutrophil proteases cathepsin g (ctsg) and elastase (ela ), and the monocyte/macrophage serine esterase, carboxylesterase (ces ). expressions of defa , b, , , ctsg and ela were signifi cantly higher in neutrophilic asthma and expression of ces was significantly higher in mixed granulocytic asthma. microarray results of the α-defensins and neutrophil proteases were successfully validated using realtime pcr. conclusions there is systemic up-regulation of α-defensins and neutrophil proteases in neutrophilic asthma, and these molecules play an important role in neutrophil activation and migration. systemic activation of neutrophils is an important feature involved in the pathogenesis of neutrophilic asthma, which is signifi cantly different to other asthma phenotypes. supported by hmri and xstrata coal; the university of newcastle. confl ict of interest no. airway mucus hypersecretion is an important cause of morbidity and mortality in asthmatic patients. increases in goblet cell number and their secretions are likely to contribute to airfl ow obstruction in asthma. here, we take advantage of an established sheep model of asthma to investigate the association between allergen exposure and goblet cell activity. methods eight allergic sheep (high house dust mite (hdm)-specifi c serum ige) received weekly intra-lung challenges of hdm to the right caudal lobe, and weekly intra-lung challenges of hdm followed by weeks without allergen exposure to the left caudal lobe, with the right medial lobe serving as an untreated internal control. a separate group of sheep were also used as untreated controls. biopsy samples of segmental bronchi tissue were collected from the different lung lobes for histological analysis at and days post-hdm challenge. results the percentage of goblet cells, with respect to epithelial cells, signifi cantly increases following chronic challenge with hdm ( % hdm vs. % control p < . ). goblet cell numbers did not decline in lung lobes after a -week cessation of allergen challenges. goblet cell degranulation is significantly increased day following challenge with allergen, but returns to control levels by days post-allergen challenge ( % day vs. % control p < . ). furthermore, degranulation is increased in both the rested and internal control lobes day following allergen challenge of the right caudal lobe. conclusions in this sheep model of chronic asthma, repeated allergen challenges induces goblet cell hyperplasia which persists even after long-term withdrawal of allergen. additionally, exposure to allergen in one lobe induces goblet cell degranulation in both challenged and unchallenged lobes, suggesting neural mechanisms may be operating in this model. confl ict of interest no. the thickness of the airway smooth muscle (asm) layer is related to severity but not duration of asthma or age (james erj; : ) . it is unknown if the constituents of the asm layer change with age. aim to investigate the relation of mean asm cell volume (v c ), total number of cells per mm of airway (n l ) and fractions of asm (f asm ) and extracellular matrix (f ecm ) within the asm layer with age and age at onset of asthma. methods post-mortem tissues from control subjects (c n = ); non-fatal (nfa n = ) and fatal (fa n = ) cases of asthma were used. the volume density (n v ) of asm cell nuclei was estimated on μm transverse airway sections (haematoxylin) and mean cell volume (v c = /n v ) was calculated, correcting for the volume fraction of asm within the asm layer. f asm and f ecm were estimated on . -μm thick sections of the same airway (masson's trichrome). effects of age on asm cell parameters and tissue volume fractions were tested using general linear models, correcting for sex and study centre and by comparing age at onset of asthma (< vs. > years). results table shows assessment of airway smooth muscle (asm) cell size and number requires estimates of cell volume density (n v ), volume fraction of muscle (f asm ) within the asm layer and the volume of asm per length of airway. stereological techniques have now become the accepted standard for assessing asm cell parameters, but sources of variation remain unclear. aim to assess sources of variability in the estimation of asm cell parameters and volume fractions within the asm layer. methods large and small airways from subjects with and without asthma were examined. transverse airway sections were cut at . μm and μm (masson's trichrome technique), and μm (haematoxylin) and used to estimate asm cell number and volume, and the volume fraction of muscle (f asm ) within the layer of asm. stereological assessments of the possible sources of variation in these asm layer parameters were assessed. results increased section thickness overestimated f asm by < % ( . μm), % ( μm) and % ( μm). stable variation of < % in n v occurred if high-power fi elds (hpf) were used to estimate n v . variation in the depth of muscle in thick sections of the asm layer caused up to % overestimation of n v . although the absolute area of the asm layer varied by up to %, variation of f asm was < % around the airway circumference and along the airway length. f asm differed signifi cantly between large and small airways. conclusion these results suggest that partial thickness hpfs need to be excluded and that ≥ hpf should be used to estimate asm volume density, that a single . μm section of airway can be used to estimate f asm and that asm parameters should be compared separately in large and small airways. grants nhmrc # . nominations nil. confl ict of interest nil. no signifi cant correlation was seen with age for any asm cell parameters or tissue fractions. results were similar for medium and small airways. conclusion size and number of asm cells and the volume fractions of asm and ecm within the layer of asm are not related to age. support nhmrc australia (grants # ; # ). nomination nil. . ± . . ± . . ± . . ± . fa > . ± . . ± . . ± . . ± . background asthma is characterized by excessive airway narrowing to contractile stimuli, termed airway hyper-responsiveness (ahr). changes in airway smooth muscle (asm) protein expression or mass are possible contributing mechanisms underlying ahr and have been examined using cell culture techniques. however, how these cellular changes to asm relate to airway narrowing at the level of the whole airway is unclear. we describe a new method to track changes in airway narrowing (responsiveness) in culture. methods whole airway segments (generation - ) from sheep lungs were studied prior to (fresh) and after and hours in culture in dulbecco's modifi ed eagle medium with % bovine serum albumin, % l-glutamine and antibiotics. airway narrowing was measured from the % decrease in airway volume under a fi xed transmural pressure, using a servo-controlled syringe pump and organ bath apparatus. cumulative acetylcholine dose-response curves (ach, − m − × − m) were performed to determine maximal response (e max ) and sensitivity (pd , negative log of ec ). results fresh airway segments narrowed strongly and approached closure with an e max of . % ± . (±sem) and pd of . ± . . airway narrowing responses were preserved in culture, with no signifi cant difference in maximal response or sensitivity to ach after either (e max . % ± . , pd . ± . ) or hours in culture (e max . % ± . , pd . ± . ). conclusions the present study has validated a new method allowing changes occurring at the cellular level in culture to be related to changes in airway responsiveness at the whole airway level. future studies will assess the effects of chronic infl ammation in disease on airway responsiveness. background deep inspiration (di) produces a bronchodilator response in healthy humans, but this response is impaired in asthma. reduced airway compliance in disease could impair the response to di by limiting the stretch of smooth muscle. aim to show that isolated human bronchi dilate to di in an amplitudedependent manner and that the stretch caused by di depends on airway compliance. methods bronchi were obtained following lung resection from cancer patients who had normal spirometry (n = ). lumen narrowing was measured using a servo-control system which set transmural pressure and simulated breathing movements. bronchi were contracted to carbachol (cch × − m) during tidal breathing (from to cmh o, i.e. Δ cmh o transmural pressure, . hz) and infl ated to three different amplitudes of di (Δ , or cmh o) applied following contraction. results in cch-contracted airways, all three di amplitudes produced a transient bronchodilation. increasing the di amplitude caused a greater increase in luminal volume during the di and a greater bronchodilation following the di (p < . ). cch itself cause approximately a % fall in specifi c compliance (p < . ), which was reversed by di (p < . ). for each di amplitude, the change in lumen volume during the di was positively correlated to the specifi c compliance of the bronchi before di (r > . , p < . ). conclusions isolated human bronchi show a bronchodilation response to di that is proportional to the expansion of the airway caused by the di. the amount of stretch produced by a di depends on airway wall compliance suggesting that increased airway stiffness in disease could suppress the di response by limiting the stretch of bronchi during lung infl ation. confl ict of interest none. ja douglass , , , ea yu , , br thompson , , , gg king , , mj abramson , introduction increasing asthma prevalence and changes in environmental exposure suggest that there may be a relationship between asthma and dietary intake. however, to date, few studies have examined how dietary intakes of asthmatics differ from a healthy population. aim to measure and compare the dietary intakes of adults with stable asthma and healthy controls. methods in a cross-sectional study, dietary intakes calculated from a item food frequency questionnaire (ffq) of adults with stable asthma (n = , age years ± (sd)) were compared with intakes of healthy controls (n = , age years ± (sd)) matched for age and body mass index (bmi). spirometry, airway responsiveness to hypertonic saline, and induced sputum cell counts were also measured. results subjects with severe persistent asthma (n = ) had signifi cantly higher total fat intake than healthy controls ( ± (sem) versus ± (sem) g/day p = . ) and signifi cantly lower fi bre intakes ( ± (sem) versus ± (sem) g/day p = . ). lower fi bre intake in asthmatic subjects (n = ) was associated with lower %predicted fev (r = . , p = . ), %fvc (r = . , p = . ) and fev /fvc (r = . , p = . ). higher fat intake and lower fi bre intake were associated with higher absolute concentrations of sputum eosinophils (r = . , p = < . , n = ). conclusions subjects with severe persistent asthma have an eating pattern of lower diet quality with higher intakes of fat and lower intakes of fi bre than healthy controls, which is related to lower lung function and increased airway infl ammation. factors leading to altered dietary intake in severe asthma require further investigation. methods a randomized, placebo-controlled, single-blinded trial of tailored asthma education including device technique and utilizing pact to address patients' concerns versus brochure-only information for asthma patients over age . measurements of lung function, asthma control (acq), asthma related quality of life (aqol), medication use and adherence score (adh) were obtained at baseline, and months using standard, validated questionnaires. results sixty-fi ve participants ( f m, mean age ± . ) were randomized to the intervention group and ( f m, mean age ± . ) to the control. there were no statistically signifi cant differences between the groups' demographics or baseline measurements. a wilcoxon signed ranks test used to compare median pair ranking at baseline and months post-intervention revealed a signifi cant improvement in the active, but not the brochure-only information group at months in: acq mean ± sd = . ± . vs. . ± . (p = . ). aqol mean ± sd = . ± . vs. . ± . (p = . ). adh mean ± sd = . ± . vs. . ± . (p < . ). conclusion an educational intervention including device technique and addressing the concerns of older people with asthma signifi cantly improved acq, aqol and adh scores at months post-intervention. introduction greater exposure to ultraviolet radiation (uv) may increase the risk of allergic disease, but this association has not been investigated using estimates of time spent outdoors by individuals. the aim of this study was to investigate the relationship between self-reported doctor-diagnosed asthma and/or hayfever, and time spent outdoors. methods this analysis was based on cross-sectional baseline data from a subsample of the australian and up study, comprising men and women aged - years, living in new south wales. participants were randomly selected from the australian universal health insurance database. diagnoses of asthma and/or hayfever and the number of hours spent outdoors were derived by questionnaire. in general, the odds of a diagnosis of asthma and/or hayfever decreased with increasing time spent outdoors for both women and men. for example, in women, the adjusted odds ratios for asthma with hayfever were . ( % ci: . - . ), . ( . - . ), . ( . - . ) and . ( . - . ) for - , - , - and > hours spent outdoors on weekends, respectively, compared with < hour (p trend < . ). time spent outdoors was not associated with a diagnosis of asthma alone in men. conclusions there were statistically signifi cant inverse associations between time spent outdoors and diagnoses of asthma, hayfever or asthma with hayfever, in a large population of older australians. exposure to uv may protect against the development of allergic diseases, such as asthma and hayfever. no. background allergic rhinitis (ar) and eczema are highly prevalent and females are more commonly affected than males in adulthood. although there have been extensive studies on ar and eczema in females, little is known about the effect of reproductive factors and the development of late-onset ar/ eczema. we examined potential associations between reproductive factors and ar and eczema using the tasmanian longitudinal health study (tahs) data. methods the tahs is a population-based cohort study of respiratory disease. two thousand seven hundred sixty-four ( . %) females from the original tahs participants were surveyed in using postal questionnaire which collected information on reproductive factors such as ever pregnancy, age at fi rst child birth, use of oral contraceptive pills (ocp) and age of starting using the ocp. logistic regression was used to assess the predictors of ar and eczema and all analyses were mutually adjusted. of the participants, . % (n = ) had late-onset ar and . % (n = ) had late-onset eczema. maternal and paternal atopy were signifi cantly associated with ar (p < . ). the risk of developing eczema was decreased signifi cantly with increasing age at fi rst menstruation (or: . , % ci: . - . ) and the increased age at birth of fi rst child ( . , . - . ). a decreased risk in ar was observed with the increasing number of pregnancies ( . , . - . ). however, the associations between age of starting using ocp and ar/eczema were not signifi cant. conclusion later age at start of menses and later age at fi rst pregnancy were associated with a reduced risk of eczema which may be related to hormonal dysregulation. tp- airway responsiveness at and years is associated with asthma at years introduction asthma is the most common chronic childhood disease in australia. increased airway responsiveness (ar) is associated with asthma but not all individuals with increased ar have asthma. the perth infant asthma follow-up study recruited a birth cohort of individuals who have undergone longitudinal assessments of many factors associated with childhood ar. our previous work reported an association between increased ar in infancy and asthma at and years. aim to look at the relationship of increased ar and asthma in early adulthood at different time points from birth. methods individuals were recruited from among expectant parents attending an antenatal clinic at a local metropolitan clinic. at ages , and months and again at , and years, participants underwent an assessment that included a respiratory questionnaire and determination of ar (as evidenced by dose-response slope (drs) to histamine using the rapid technique). results children were initially recruited and studied in infancy. two hundred three, , , , and children subsequently had ar assessed at , and months, , and years, respectively. there was a signifi cant relationship between drs at and years and for both asthma at years (p = . and p < . , respectively) and 'wheeze in the past year' at years (p = . and p = . , respectively). there was no significant relationship between drs in infancy and asthma at . conclusion ar at and years is associated with asthma at years. in this study, there was no signifi cant relationship between ar in infancy and asthma at years. the pcaas has found that . % of children with acute asthma presenting to the princess margaret hospital for children emergency department (pmh ed) had hrv, of which % were hrv group c. furthermore, hrvc was associated with more severe attacks. however, the prevalence of hrvc in the community is unknown. aim to test the hypothesis that hrvc would be found more often in children requiring emergency treatment for an ari than sibling controls and determine the impact of days since symptoms began on the prevalence of hrv detection in children with an acute respiratory illness (ari) and sibling controls (sibs). methods ari (n = ) had nasal samples collected on presentation to the pmh ed and sibs with symptoms of a cold (n = ), within week of ari recruitment. viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by dna sequencing for typing. results ari and sibs were % and % male, % and % asthmatic, with mean ages of . and . years, respectively. hrv +ve ari (n = , mean ± sd days of symptoms = . ± . ), hrv -ve ari (n = , . ± . ), hrv +ve sibs (n = , . ± . ) and hrv -ve sibs (n = , . ± . ). of the and hrv +ve ari and sibs, % and % had hrvc. conclusions hrvc is as common in children who have hrv but do/do not require hospital treatment. detection of hrv is more likely when the nasal sample is collected soon after the appearance of cold symptoms. support nhmrc program grant. nomination nil. introduction upper airway dysfunction may make asthma more diffi cult to control and should be suspected in asthmatics refractory to prescribed medical therapy. aim a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in healthy and asthmatic individuals. method vocal cord movement was imaged using -slice ct larynx. healthy volunteers were studied to develop and validate an analysis algorithm for quantifi cation of normal vocal cord function. further studies were then conducted in patients with diffi cult-to-treat asthma. in eight severe asthmatics with abnormal vocal cord movement, asthma outcomes were measured after speech therapy. results vocal cord movement was quantifi ed over the breathing cycle by ct using the ratio of vocal cord diameter to tracheal diameter. normal limits were calculated, validated and applied to evaluate diffi cult-to-treat asthma. vocal cord movement was abnormal with excessive narrowing in of ( %) asthmatics and severe in nine ( %) patients (abnormal > % of inspiration or expiration time). after speech therapy in a small subgroup, asthma symptoms and morbidity improved. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was achieved. this new approach has identifi ed frequent upper airway dysfunction in asthma with potential implications for disease control and treatment. aim to investigate the characteristics and mechanisms of chronic cough (cc) following acute respiratory illness from laboratory-confi rmed h n infl uenza. methods subjects who had current symptoms and had been tested for h n infl uenza by pcr assay participated in this study. twenty-one of those continued onto clinical testing. investigations to assess cough included symptom questionnaires, hypertonic saline challenge and cough monitoring. results of the participants, % tested positive for h n and % tested negative for h n . h n -infected participants were younger and predominantly female. the prevalence of post-h n cc was . %, and for non-h n infection, . %. objectively measured cough frequency was times greater; there was a -fold increase in cough refl ex sensitivity, and greater quality-of-life impairment in the participants with chronic post-infectious cough than the non-cough participants. conclusions cc was found to be relatively common, mild in severity and tending to resolution with time. the characteristics of post-h n cc were similar to other post-infectious cough and were associated with cough refl ex hypersensitivity. aim upper airway dysfunction may accompany acute severe asthma, but this has not been investigated. a novel imaging technique, dynamic -slice computerized tomography (ct), was used to examine laryngeal behaviour in acute asthma exacerbation. methods patients were studied in the emergency department or as acute inpatients following admission for an acute exacerbation of asthma. vocal cord movement was imaged by -slice ct larynx and compared to normal vocal cord movement in a healthy cohort. results vocal cord movement was abnormal with excessive narrowing during either inspiration, expiration or both in of cases ( . %) with acute severe asthma. imaging again revealed that laryngeal dysfunction characterized the movement abnormality, rather than isolated vocal cord dysfunction. radiation exposure was low and generally < milli-sievert. conclusion non-invasive ct larynx quantifi cation of vocal cord movement was effectively achieved in acute severe asthma. we identifi ed frequent upper airway dysfunction in acute severe asthma suggesting that treatment of upper airway obstruction (e.g. using bipap) may be merited during asthma exacerbation. aim to determine whether eicosanoids could alter the deposition of extracellular matrix (ecm) proteins and cytokine release from human airway cells. methods airway smooth muscle cells (asm), fi broblasts and epithelial cells were stimulated with leukotrienes b , c , d , e and the prostaglandins e , d , f α and the pgi analogue mre- . after hours, culture medium was collected and il- and il- production and cell deposited ecm proteins tenascin c, fi bronectin and perlecan were assessed by elisa. to determine whether eicosanoids infl uenced cell proliferation, manual counting of cells in the experiments were carried out before and after stimulation. results neither leukotrienes or prostanoids altered cell proliferation after days of stimulation (n > ). leukotrienes had no effect on ecm protein deposition or cytokine release from asm or fi broblasts (n > ). leukotrienes did not alter either parameters in epithelial cells except leukotriene d , which increased tenascin c deposition (n = , p < . ). prostanoids induced il- and il- and other various changes in asm and fi broblasts (n > , p < . ) (see below). introduction the function of asthmatic airway epithelium is disrupted facilitating immune and infl ammatory responses resulting in epithelial damage. human rhinovirus (hrv) causes asthma exacerbations in children; however, paucity exists on how it affects barrier function. this study assessed how hrv infection affects epithelial barrier function and integrity in healthy and asthmatic epithelium. methods adult balb/c mice were intranasally infected with hrv- b and followed for days. tight junction (tj) expression was assessed using immunohistochemistry (ihc) and western blot analysis. primary airway epithelial cells from healthy and asthmatic children were assessed for tj gene and protein expression by qpcr and ihc, respectively. results occludin and zonal occludin- (zo- ) expression was lost and sustained in mice infected with hrv- b however was not observed in shaminjected mice. asthmatic airway epithelial cells were found to exhibit elevated basal gene expression levels of tjs (zo- , occludin and plakophilin- (pkp- )) but markedly lower corresponding protein levels. conclusion hrv- b compromises barrier function in vivo through sustained loss of tj proteins. the marked decreased expression of tj proteins in paediatric asthmatic epithelium may contribute towards increased susceptibility to viral infections. disparity between gene and protein tj expression could indicate either post-transcriptional regulation or compensatory effects by other tj proteins and requires further study. supported by asthma foundation wa; nhmrc. confl ict of interest none. conclusion leukotrienes alone did not affect the ecm proteins and cytokines assessed in this study. prostanoids decreased ecm protein deposition whilst increasing cytokine release without affecting cell proliferation. this study shows that prostanoids may have a more pronounced role on direct ecm remodelling than leukotrienes in airway cells. supported by merck. background toll-like receptor (tlr) is an innate immune receptor involved in the initial detection of pathogen-associated molecular patterns. the effect of ageing and chronic obstructive pulmonary disease (copd) on tlr responses and the impact of these innate immune responses in copd pathogenesis remain unclear. hypothesis expression and activity of tlr on peripheral blood mononuclear cells (pbmcs) is increased with healthy ageing and further increased in copd. methods pbmcs from healthy controls < years and > years; and participants with copd (n = per group) were cultured with or without pam c ys k (tlr agonist). cells and supernatants were collected at hours and protein (cytometric bead array or fl ow cytometry) and gene (real time pcr) expression was examined. results tlr activation led to increased release of interleukin (il)- , , β, and tumor necrosis factor (tnf)-α. tlr gene expression was increased with stimulation; however, cell surface receptor levels were unchanged. there was no difference in the level of tlr between the groups. in older people, tlr activation resulted in less il- β and tnf-α release, but similar release of il- and il- . similar results were seen in copd. at baseline in copd, there was up-regulation of tnf-α gene expression compared to the older healthy group; however, the tlr cytokine response did not differ between the groups. conclusion healthy ageing is characterized by an impaired systemic proinfl ammatory cytokine response to tlr -mediated innate immune activation. this effect persists in copd and is selective in the cytokine pathways involved. these altered infl ammatory mechanisms may affect responses to infection and injury impacting disease pathogenesis and warrant further evaluation. aim to investigate whether the inhibition of matrix metalloproteinase- (mmp- ) by a non-selective mmp inhibitor (doxycycline) and the specifi c mmp- inhibitor i (olic acid) can regulate cellular migration of tsc -null mouse embryonic fi broblasts (mefs), which act as a model for lymphangioleiomyomatosis (lam) cells, as compared to wild-type mefs. methods wild-type (tsc -positive) and tsc -null mefs were treated with diluent, doxycycline ( . pg/ml- μg/ml) or olic acid ( . - μm) for hours. mmp- levels were assessed by zymography and elisa. cell migration for hours was measured using a transwell migration assay. results under basal conditions, mmp- release and cellular migration was . -fold and . -fold higher, respectively, in tsc -null mefs compared to tsc -positive mefs (mmp- release, tsc -null (n = ) and tsc -positive (n = ), p < . ; cell migration, tsc -null (n = ) and tsc -positive (n = ), p < . ). mmp- release was reduced in tsc -null mefs after -hour treatment with doxycycline ( and μg/ml, n = , p < . ) and with olic acid ( - μm, n = , p < . ). treatment with doxycycline ( pg/ml- μg/ml, n = , p < . ) or olic acid ( - μm, n = , p < . ) also signifi cantly reduced cell migration of tsc -null mefs. copd is a leading cause of death worldwide. treatments are limited and restricted to symptomatic care. there is an urgent need for new treatment options targeting the infl ammation. tissue damage in copd is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. lung infl ammation and macrophage function involves the bioactive sphingolipid sphingosine -phosphate (s p). multiple studies have showed the involvement of these components in infl ammation. methods we investigated lung tissue samples from patients (copd or non copd controls) undergoing curative lobectomy for lung cancer. we analysed the mrna expression profi le, the sphingosine-kinase (sphk) protein activity and the localization and expression of individual proteins. results we show in this study for the fi rst time a comprehensive expression profi le of all synthesizing enzymes, receptors and degrading enzymes in the human lung. correlations between receptor subtypes, degrading enzymes and between s p receptor subtype were detected. multivariance anova showed that in copd, the relative mrna expression of s p receptor subtype was reduced. conclusion the correlations between receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. s pr is expressed on dendritic and nk cells which are reduced under conditions of copd. therefore, our fi ndings of reduced s pr in copd may provide a novel target for pharmacotherapy. lung cancer is responsible for more cancer-related deaths than colon, breast and prostate cancers combined. in patients with copd and/or lung cancer, we have shown a reduction in lung and airway macrophage function, evident by a reduced ability to phagocytose apoptotic airway epithelial cells and neutrophils. the potential for lung cancer cells to directly inhibit this function (a potential immune evasion mechanism) has not been investigated. background kinins have been implicated in airway lung diseases such as asthma and lung cancer by regulating infl ammation, cell proliferation and migration. the effect of kinins is mediated through the binding of two receptors, kinin b and b receptors (b r and b r). a novel b r splice variant (sv) resulting in a shorter ' untranslated region (utr) was identifi ed in cultured airway epithelial and fi broblasts as well as in lung carcinoma tissue and leukocytes. this study aims to characterize the functional role of the novel b r sv in mrna stability, translation effi ciency and receptor expression in cultured airway epithelial cells. methods stability of b r sv was determined by measuring b r mrna levels over time in h cells after actinomycin d treatment. translational effi ciency of wt and sv 'utr was determined by measuring luciferase activity in transfected h cells. expression of wt and sv transcripts through q-rtpcr were compared in cells treated with a b r-specifi c agonist dakd. cell-surface receptor expression post-agonist stimulation was quantifi ed using facs. results mrna stability studies indicated that b r sv was ≈ % less stable than the wt transcript in h cells suggesting a stabilizing element 'utr. translation effi ciency of sv was no different to wt b r. dakd stimulation increased both wt and sv transcripts early in the time course, although the peak expression of wt and sv differed at hours and hours, respectively. dakd stimulated cells showed two phases of receptor expression, ( ) decrease of cell surface receptor up to . hours post-stimulation; ( ) increase in cell surface b r after . hours. conclusion this study has identifi ed a novel regulatory mechanism of b r expression through the production of a sv that alters the 'utr. the translation effi ciency of b r is not affected, but the sv was less stable than the wt in h cells and may play a role in allowing quicker changes in transcription. agonist-induced up-regulation of transcripts in a time-dependent manner may be important in maintaining a chronic response during infl ammation. circulating lymphocytes are increasingly used as a surrogate cell type to refl ect changes in adrβ density elsewhere in the body, particularly the respiratory system. however, adrβ density is non-uniform among lymphocyte subsets and it is unclear if, and the degree to which, adrβ density varies between individuals. aim to assess the extent of variability in adrβ density on human peripheral blood mononuclear cells (pbmc) including lymphocytes and monocytes. method pbmc were isolated from blood of healthy subjects by density gradient centrifugation with ficoll-paque. cell surface and total adrβ of intact and permeabilized lymphocytes (cd +) and monocytes (cd +) were measured using anti-adrβ via facs. geometric mean fl uorescence (gmf) was used as the indices for adrβ density per cell. result surface adrβ -gmf increased by . -and . -folds over negative controls for lymphocytes and monocytes, respectively. magnitude of foldchange was not signifi cantly different between these cells (p = . ), but the distribution of gmf intensity between samples suggests greater variability in adrβ density in lymphocytes versus monocytes (p = . ). proportion of cells-stained adrβ -positive was signifi cantly higher in monocytes versus lymphocytes ( . ± . % vs. . ± . %, p = . ). total adrβ -gmf increased by . ± . and . ± . -folds for lymphocytes and monocytes, respectively (p > . ). proportion of adrβ -positively stained cells were similar between samples (lymphocytes %, monocytes %, p = . ), but greater variability was observed for lymphocytes (range - %) versus monocytes ( - %). conclusions despite similarities in surface and total adrβ density, lymphocytes display greater inter-subject variability compared with monocytes. this will have implication in experimental designs and interpretation of changes in adrβ density in studies using human pbmc as an alternative to primary cells from the organ of interest. confl ict of interest no. pge plays a protective role in asthma by inhibiting airway infl ammation. it is predominantly produced by epithelial cells in response to pro-infl ammatory stimuli and acts as an autocrine and paracrine mediator. on the contrary, il- β is a highly potent cytokine that induces many pro-infl ammatory effects in the human airway including activation of the human lung epithelium which promotes production of pro-infl ammatory cytokines and chemokines. airway epithelial cells express all four known pge (e prostanoid (ep) receptors, but mechanisms underlying the regulation of expression of ep receptors in human lung epithelial cells have remained elusive. therefore, we investigated whether pge , an endogenous protective mechanism of the airways, can modulate il- β infl uence on ep receptor expression in human epithelial cells. methods ep receptor mrna and protein expression was quantifi ed in -hbe cells at basal levels and following stimulation with il- β or pge alone, or simultaneously, using real time rt pcr and facs analysis, respectively. results pge up-regulates all four ep receptors at mrna level, while il- β up-regulates ep , ep and ep and does not infl uence expression of ep . at protein level, preliminary results show transient increase of ep receptors in the presence of pge , while il- β down-regulates this receptor. ep and ep are up-regulated following stimulation with both stimuli. importantly, antiinfl ammatory ep receptor is up-regulated only in the presence of pge . conclusion we show for the fi rst time that pge may infl uence expression of its own receptors and oppose the effect of il- β in human lung epithelial cells. this may in turn alter pge production and autocrine activation with potential implication on the function of epithelial cells, which is important in modulation of immune response in asthma and lung infl ammatory diseases. nomination nil. confl ict of interest no. the burden of obstructive lung disease (bold) study is an international study designed to measure the prevalence, risk factors and burden of copd. data collection using the bold protocol has been undertaken at eight sites with inclusion of urban, rural, coastal and inland regions of australia. methods a random sample of adults aged ≥ years was identifi ed. information on respiratory symptoms and diagnosed copd were collected by questionnaire. post-bronchodilator fev and fvc were used to defi ne gold stage. the (un-weighted) prevalence rates are presented by age groups and sex. results s timmins , , , , g king , , , , c salome , , , r schoeffel , , , c walsh , , the extent of emphysema could increase ventilation heterogeneity independently of its effects on airway narrowing. the aim of this study was to examine the relationship between emphysema extent on computed tomography scans (ct), and airway narrowing and ventilation distribution in copd. methods subjects with copd underwent ct scanning, spirometry, dlco and nitrogen washout by single and multiple breath techniques. closing capacity (cc/tlc%), slope of phase iii (Δphase iii ) and indices of ventilation distribution conductive (scond) and diffusion-dependent airways (sacin) were derived from washouts. helical ct scans were performed at tlc. emphysema extent was measured as low attenuation areas < − hu using osirix program, expressed as % of ct total lung volume. results subjects were of mean (range) age years ( - ), bmi . ( . - . ), fev of ( - %) %predicted and dlco of ( - ) %predicted. emphysema extent was . % ( . - . ). geometric mean (ci) Δphase iii was . ( . - . ), sacin was increased at . l − ( . - . ) and cc/tlc% was % ( - ). emphysema extent correlated with fev / fvc (r = − . , p = . ), dlco (r = − . , p < . ), bmi (r = . , p = . ), Δphase iii (r = . , p = . ), and sacin (r = . p = . ). in multiple regression analysis, emphysema extent was predicted by fev /fvc and Δphase iii (model r = . , p = . ). conclusions the extent of emphysema increases the heterogeneity of ventilation independently of any effects on overall airway narrowing. supported by australian lung foundation webster memorial award, crcaa. conclusions self-reported wheeze in the last months is very common in adults over years. in the younger age group ( - years), many people with wheeze did not have airfl ow obstruction or reversible spirometry at the time of test. aim to determine whether there is any association between change in fev among copd patients and ambient ultrafi ne particle number concentrations in melbourne. methods participants with mild to moderate copd were asked to measure their fev using a portable electronic spirometer (piko) two times a day (morning and evening) for consecutive days. the same procedure was repeated on average months later. ambient ultrafi ne (diameter < . μm) particle number concentrations were measured for the same period using an ultrafi ne condensation particle counter and micro-orifi ce uniform deposit impactor. results aim to examine the implementation of, and barriers and enablers to, six high-evidence recommendations for copd management, in copd hospital inpatients. method observational, mixed methods study in consecutive copd patients admitted to a tertiary hospital. demographic, disease and admission characteristics are recorded. implementation (or not) of smoking cessation, pulmonary rehabilitation, long-term oxygen use if hypoxaemic, medication use, vaccinations and plans for future exacerbations are determined from medical records and patient interviews. interviews with medical offi cers examine their perspectives on recommendation implementation. of pilot data in copd patients (mean (sd) age ( ) years, length of stay ( ) days), were current smokers and had severe copd ( moderate). highest levels of implementation were fl u vaccination (completed by gps, n = ), medication (but not spacer) use, and oxygen use if hypoxaemic (investigated and implemented in all suitable, n = ). pulmonary rehabilitation was discussed with half of the patients, but only severe patients with long length of stay accepted further rehabilitation. exacerbation plans were in place for patient, and newly initiated in patients. doctor interviews (n = ) confi rmed pulmonary rehabilitation was considered mostly for severely unwell patients, and use of exacerbation plans was inconsistent. conclusion pilot data suggest pulmonary rehabilitation is offered and accepted by a small subset of copd patients. findings from this pilot will inform planned larger observational studies, and in turn, experimental studies to improve copd care. high-and extreme high-risk interventions were found by panel ( - . % extreme and . - . % high-risk interventions) and patients' respiratory physicians ( % extreme and % high-risk interventions). additionally, clinical pharmacist involvement was associated with many benefi ts such as: improvement in medication compliance, high level of patient satisfaction and identifi cation of patients with issues in medication knowledge. conclusion clinical pharmacist interventions were estimated to prevent extreme and high risks that might happen due to drug-related problems. clinical pharmacy consultation was associated with positive impact on other important measured outcomes. aerobic exercise training in the form of supervised -minute walks ( mw) reduces exertional dyspnoea in patients with copd. mw goal ( mwg) distances, aiming for a training effect, are generated from a baseline submaximal test ( -minute walk ( mwd), where wg = . × mwd/ × . aim to compare mwg with actual initial mw achieved and to examine the predictors of mwg achievers (ga). methods retrospective review of patients, % male, age ± years (mean ± sd), fev ± %predicted, who completed pulmonary rehabilitation (pr). patients were assessed at baseline and post-completion of pr. initial mwg was calculated from the best of two mwd at initial assessment and ga were defi ned as patients who achieved their mwg at their fi rst visit to pr. results for the group, there was a statistically signifi cant but not clinically signifi cant difference between mwg and actual mw achieved ( ± m vs. ± m, p < . , paired t-test). the patients ( %) who achieved their mwg exceeded the goal by ± m, whereas the patients who did not achieve their mwg fell short by ± m. there was no signifi cant difference between ga and non-ga in age or lung function, but ga had a higher initial mwd, with fewer rests, lower dyspnoea score and lower hr at start and fi nish (p < . , unpaired t-test). ga were also more likely to have a clinically signifi cant response to pr, measured by mwd, compared with non-ga (mean change m vs. m, p < . , chi-square). conclusion mw goals as currently calculated either signifi cantly underestimate or overestimate actual mw achieved. it may be that in non-ga, the mwd is functioning as a true maximal test and these are a group of patients who are truly ventilatory-limited, rather than deconditioned. the receptor for advanced glycation end products (rage) is a key candidate for promoting a self-perpetuating cycle of infl ammation and thereby is a major contributor to numerous chronic disease states. the potential of rage to function as a switch converting a transient infl ammatory response such as one generated by cigarette smoke to sustained cellular dysfunction allows it to act as a mediator for ongoing infl ammation in chronic obstructive pulmonary disease (copd). although the molecular mechanisms regulating rage expression have not been fully elucidated, altered rage activity arises from polymorphisms within the rage gene and its promoter. three polymorphisms in the rage promoter (− t/a, − t/c and a bp deletion from − to − ) increase transcriptional activity and rage expression. the rage g s allele results in an increased ligand-binding affi nity and activation of the infl ammatory mediators with subsequent up-regulation of infl ammatory response. the aim of this pilot cross-sectional study was to investigate the relationship between three known rage polymorphisms (− t/a, bp deletion, g s) and copd and disease severity. methods genomic dna was isolated from peripheral blood lymphocytes. pcr and taqman assays were used to genotype the three rage polymorphisms in copd patients, healthy non-smokers and healthy smokers. fev was measured in all subjects. disease severity was defi ned using gold guidelines. results there was no statistically signifi cant association between bp deletion and copd (p = . ), − t > a and copd (p = . ), g s and copd (p = . ). conclusion no association was found between the − t > a, bp deletion and g s polymorphisms and copd, disease severity or fev introduction the receptor for advanced glycation end products (rage) mediates neutrophil traffi cking and is implicated in the pathogenesis of chronic airways disease. we determined whether changes in airway and systemic levels of soluble rage (which acts as a receptor decoy to limit rage activation) and rage ligands are related to neutrophilic infl ammation in asthma and copd. methods bronchial lavage (bl) fl uid from subjects with moderate-severe persistent asthma or copd, and healthy controls were analysed for neutrophils, total srage (cleaved and secreted), secreted srage (esrage) and the rage ligands hmgb and serum amyloid a (saa). systemic levels srage and esrage were also determined in asthmatic and copd subjects. aims increased numbers of neutrophils are found in the lungs of copd patients, which contribute to airway infl ammation. while cigarette smoke exposure is the major risk factor for copd, it is unclear how cigarette smoke modifi es neutrophil function and activity. this study aimed to assess the effect of cigarette smoke extract (cse) on neutrophils in an in vitro model. methods neutrophils were isolated from peripheral blood donated by volunteers using percoll density gradient centrifugation. neutrophils were seeded in well plates ( cells/well), exposed to different concentrations of cse ( %, %) and monitored at , and hours. at each time point, viability of neutrophils was measured by trypan blue exclusion and supernatant was collected for measurement of cxcl release by elisa (r&d systems conclusions in neutrophils exposed to cse, viability is maintained and cxcl release increases with increasing dose of cse. we conclude that cigarette smoke stimulates an infl ammatory response by neutrophils, which would contribute to the infl ammatory burden in the airways in copd. introduction factor viii (f ) and collagen iv (c ) antibodies are used for quantifying vessels in tissue sections. we compared these two antibodies for vessels staining in bronchial biopsies (bb) in copd. methods bb from healthy non-smokers (h-n) and copd subjects were stained for both antibodies. number, area and mean vascular size (mvs) (surface area/vessel number) of vessels in the lamina propria (lp) to the depth of μm were measured and compared between the two antibodies and are reported as median (range). results number of vessels was not signifi cantly different between the two methods of staining. in copd and h-n, vascular area (μm /μm of lp × ) stained with f was less than that with c ( . ( . - ) vs. ( - . ), p < . and . ( . - . ) vs. . ( . - . ), p < . introduction previous studies have shown that c-reactive protein levels increase at the onset of some copd exacerbations; however, there is limited data on the normal fl uctuation in crp levels in stable patients. aim to investigate within patient variation in crp levels to determine the magnitude of normal day-to-day fl uctuations in stable patients and the correlation with patients' perception of symptom severity. methods early morning crp levels were measured on days , and from patients from the melbourne copd cohort (gold category ii-iv) who identifi ed themselves as stable. patients recorded daily symptom scores including: borg dyspnoea scale at rest, severity of wheeze, cough, dyspnoea, change in sputum colour or volume, night-time waking and the presence of viral symptoms. crp levels were measured by the clinical pathology service and using a point-of care device. variation in crp levels in stable copd and correlation between change in crp levels and symptoms were analysed. aim patient-completed diaries monitoring changes in key symptoms in copd are often used to recognize acute exacerbations (ae) both to prompt additional treatment and monitor treatment effi cacy. we assessed diary compliance and the predictive value of major symptoms of aes which required hospital attendance. methods inpatients recruited during an ae of copd completed daily paper or web-based diaries for months, recording changes from their stable state for: breathlessness, cough, sputum, subjective 'wellness', physical activity and use of reliever ( -point scale, mid-pt = no change). the predictive value of current and lagged symptom scores was compared for each and between symptoms. diagnostic accuracy was assessed by area under the curve (auc) and at specifi c cut-points. in participants ( m, f) with mean age ± and mean fev % predicted ± , there were such aes involving patients. duration of diary keeping was shorter with lower education attainment (p = . ), but compliance did not vary for other demographic or clinical factors. daily compliance while diaries were being kept was %. excluding the current day, the best predictor was the distributed lag score over days, sputum changes giving the strongest signal; relative risk . ( % ci . to . ) with most of the signal in the days prior to the ae. little was gained by combining symptoms. the predictive value was moderate auc = . . conclusions compliance with symptom diaries in severe copd is surprisingly good. however, with only a weak signal for an impending ae requiring hospital attendance up to hours before and for lagged symptom scores over days before, with low positive predictive values, the utility of keeping daily symptom diaries for raising alerts for impending severe aes in copd is questionable. results seven studies with inpatient participants were identifi ed; published as abstracts for which data were not available did not contribute to meta-analyses. no study specifi ed diagnostic criteria for copd and only one specifi ed ae criteria. short course treatment varied between - days and longer duration - days; studies used oral prednisolone (dose mg, studies, tapered dose) and studies used intravenous scs treatment. mean ages of participants ranged from to years. primary outcomes: likelihood of treatment failure did not differ by duration of treatment (odds ratio . ; % ci . to . ) ( studies, n = ). fev did not differ signifi cantly when measured up to days (mean difference (md) − . l; % ci − . to . ) or after days (md − . l; % ci − . to . ) ( studies, n = ). secondary outcomes: limited data ( study) precluded meta-analysis for readmission or mortality. the likelihood of an adverse event ( studies, n = ) was not signifi cantly lower for shorter scs (or . ; % ci . to . ). conclusions we found no signifi cant differences between short (≤ days) and longer (> days) corticosteroid therapy for ae of copd. this has implications for clinical practice and may reduce adverse effects for patients, shorten hospital admissions and reduce costs, but more studies are needed to confi rm these fi ndings. aim to explore factors which infl uence the self-management of exacerbations in patients with copd. methods a pilot cross-sectional study was undertaken to assess patients' compliance with their action plan and their action taken prior to an admission. patients were interviewed during an admission to hospital for exacerbation of copd. the effect of pulmonary rehabilitation on patients' knowledge of copd was also assessed. results % of patients were provided with a written action plan, and % with a verbal action plan. in response to an exacerbation, more than % of the patients stated that they used their action plan. however, where action plans were not adequately utilized, patients delayed seeking medical attention and failed to initiate oral prednisolone and antibiotics during an exacerbation despite being prescribed an emergency supply of these medications. pulmonary rehabilitation had a positive outcome towards enhancing the patients' knowledge of copd. clinical pharmacists have limited involvement in terms of copd and smoking cessation education. conclusion the need to offer a thorough self-management program along with providing a more comprehensive written action plan will encourage patients to start early treatment and follow their action plans. encouraging collaboration between the hcp and patients encourages self-management through discussing and agreeing on goals of treatment and developing a personalized written action plan. context dyspnoea is a common symptom in copd and increases during exacerbations. when respiratory failure supervenes, and assisted ventilation is required, non-invasive ventilation (niv) is the treatment of choice. objective to determine if niv relieves dyspnoea in inpatients with acute respiratory failure due to exacerbations of copd. data sources english language randomized controlled trials (rcts) published prior to august were identifi ed using medline, embase, cinahl, psychinfo and pubmed. additional studies were identifi ed by reviewing the reference list of included studies. search terms included niv, nippv, nppv, bilevel cpap, bipap, artifi cial ventilation, copd and randomized controlled trial. study selection rcts comparing usual medical care (umc) to umc plus niv and measuring dyspnoea at relevant time points were included. abstracts for potentially relevant articles were extracted by one author. these were assessed by a second author to ensure inclusion criteria were met. articles were reviewed to determine if dyspnoea was measured and appropriate statistical analysis reported. the search yielded individual articles. four articles met the review criteria. three articles fi nd that niv relieved dyspnoea to a statistically signifi cant level and two suggested that the relief of dyspnoea is clinically signifi cant. discussion in spite of the common use of niv to relieve dyspnoea, little work has analysed effi cacy in terms of this patient-reported outcome. while our results may suggest niv relieves dyspnoea, reporting or methodological fl aws in several articles limit the strength of the conclusions that may be drawn. these limitations make the conclusion that niv relieves dyspnoea contentious. conclusion despite over two decades of studies investigating niv, the therapeutic impact on breathlessness is poorly described. understanding the impact of niv on patient-reported outcomes is of critical importance in clinical care. confl ict of interest none. introduction in mice, the most direct lung dosing method delivers the agents directly into the trachea. for our cystic fi brosis gene-therapy studies, we deliver fl uids -an airway pretreatment followed by a lentiviral vector -directly into the mouse trachea to target conducting airways. despite using standardized delivery techniques, we see substantial variability in the amount and location of gene transfer. aim the aim of this experiment was to use synchrotron x-ray imaging to track the dynamics of fl uid doses delivered into the live mouse trachea. methods four nembutal anaesthetized c bl/ mice were imaged on the bl b beamline at the spring- synchrotron. mice were intubated and ventilated at br/min with image captured per breath. after minute of baseline, a -μl sample of iodine-based contrast fl uid (a surrogate for our airway pretreatment or gene-vector) was delivered over seconds. following minutes of data collection, an additional μl bolus was delivered over . seconds. image capture continued for a further minutes. frame differencing was used to reveal fl uid motion. results substantial dose losses may occur upon delivery into mouse trachea via immediate retrograde fl uid motion. the speed of bolus delivery into lung may also infl uence the relative targeting of conducting airways and deep lung. introduction use of effi cient nebulizers can enhance the quality of life of cf patients by reducing the treatment time and improving drug delivery effi ciency. the aim of this study was to determine which commonly recommended nebulizer was optimal for delivery of the most commonly used therapies to cf. methods seventeen children with cf ( - years) were recruited. delivery of three commonly used cf therapies ( % hypertonic saline ( ml, . g/ ml), tobramycin ( ml, mg/ml) and pulmozyme ( . ml, mg/ml)) by two vibrating membrane nebulizers, the eflow rapid and the aeroneb go, and a jet nebulizer lc sprint junior with pariboy sx ( . l/min) were tested. for each drug-nebulizer combination (in random order), each child was asked to inhale through an inspiratory fi lter, and drug delivery to the fi lter was measured. pulmozyme was quantifi ed using an enzymatic activity assay, tobramycin was measured using hplc and hypertonic saline was measured using conductivity. total nebulization time was recorded. the results showed that there was no difference in the amount of drug delivered to patients when the nebulizers were compared for all three therapies (p > . ). however, the nebulization time for the eflow rapid was signifi cantly shorter than that for the aeroneb go and lc sprint junior. similarly, the nebulization time for aeroneb go was shorter than that for the lc sprint junior (p > . ) for all therapies). conclusion overall, there were no signifi cant differences between nebulizers in delivered dose for three forms of cf therapy, due to inter-patient variability. despite this, both vibrating membrane nebulizers had shorter nebulization times than the lc sprint junior, with the eflow rapid delivering drug in the shortest time. confl ict of interest nil. introduction as the life expectancy of patients with cystic fi brosis (cf) increases, treatment-related morbidity is increasingly recognized. totally implantable venous access devices (tivads) offer reliable long-term central venous access but are associated with recognized complications including venous thrombosis. superior vena cava syndrome (svcs) however has been rarely reported in this setting. we report a single cf centre's experience of svcs associated with tivads. methods retrospective review of episodes of svcs in patients with cf and a tivad attending the adult cf centre, prince charles hospital, queensland. results between february and december , fi ve episodes of svcs occurred in patients with tivads from a clinic population of patients. all of the affected patients were female, with moderately severe lung disease (mean fev predicted . %). no patients had a recognized thrombophilia. four tivads were inserted at a centre different to our own, three were on oestrogen-based contraception, and two suffered with dehydration at presentation. svcs treatment consisted of anticoagulation ( ), line removal ( ), angioplasty ( ), thrombolysis ( ) noninvasive bioluminescence imaging has allowed for rapid in vivo quantifi cation of long-lasting gene transfer in experimental animals. we are testing the longevity of a single nasal delivery of our lentiviral (lv) gene transfer system in mouse airways. methods normal (c bl/ ) and cystic fi brosis (cf) mice received a nasal bolus of lysophosphatidylcholine (lpc) or a control (pbs) pretreatment hour prior to delivery of a lv vector containing the reporter-gene luciferase (lv-luc). another control group received lpc hour prior to an empty vector (lv-mt). bioluminescence was measured at week, , , , , , , , and months post-lv dosing to assess gene transfer. results normal mice: mice that received lpc/lv-luc treatment had significantly greater gene transfer compared to the two control groups at all time points (p < . , rm anova). no luminescence was detected in mice treated with lpc/lv-mt. unexpectedly, luciferase activity was also detected in the lung. there was no difference in lung luminescence between the lpc and pbs pretreated mice that received lv-luc. cf mice: a statistically signifi cant increase in nasal luminescence persisted for up to months following lpc/ lv-luc (p < . , rm anova). similar to normal mice, there was no statistical difference in lung luminescence between mice that received lpc and pbs lv-luc. conclusions lentiviral luciferase gene expression was signifi cantly improved in mouse nasal airways using lpc pretreatment in both strains. however, the longevity of transduction was reduced in cf mice, which may, in part, be due to reduced animal numbers at the later time points tested. supported by nh&mrc. background the nintendo-wii® facilitates exercise-based programs that may be considered novel, fun and potentially motivating. objective exercise outcomes using the wii have yet to be reported in the cystic fi brosis (cf) adult population. aim to investigate nintendo-wii® exercise training compared with standard exercise in adult cf patients whilst hospitalized for treatment of a pulmonary exacerbation. methods a within-subjects, randomized cross-over study. adult cf participants received two -minute exercise treatment sessions within a -hour period, at least day apart, during the last days of hospitalization. wii exercise consisted interval training with games such as boxing, dancing and track exercises. standard exercise consisted of interval training on treadmill or cycle ergometer at - % of heart rate maximum. results participants completed the study (mean (sd) age ( ) years, % females), with a mean fev % of ( )%. during exercise, no difference was found between groups in average heart rate (p = . ), oxygen desaturation (p = . ), borg rate of perceived exertion (p = . ) or modifi ed borg for dyspnoea (p = . ). on vas ( - ), participants reported the wii program to be more enjoyable (p < . ) and less fatiguing (p = . ). participants rated both exercise sessions as equally effective (p = . ). conclusions this study suggests that a nintendo-wii® exercise session provides an equivalent cardiovascular demand to a standard exercise session in an inpatient adult cf population. greater enjoyment levels and lower fatigue levels reported during nintendo-wii® training may have a positive infl uence on adherence to exercise. further study into the long-term effects of nintendo-wii® training needs to be undertaken. confl ict of interest nil. introduction ion transport is important to maintain the airway epithelial surface, as shown by the disease cystic fi brosis (cf) which is characterized by decreased clsecretion and increased na + absorption. we have previously shown that the cf airway can develop clresponses when the surface is nominally calcium free (middleton et al. ajrccm ; : - . aim to determine the effects of citrate on the nasal potential difference (npd) with and without amiloride pretreatment, and to compare these effects with other clinically relevant calcium chelators and dicarboxylic acids. methods npd was measured using standard techniques (erj ; : ) in cf and non-cf subjects. the nasal pd response to citrate, oxalate, malate, succinate and fumarate (all mm) was compared with the calcium chelators edta and egta. results citrate decreased the basal npd by ∼ mv, but in the presence of amiloride, citrate increased the pd by ∼ mv. with amiloride/low clpretreatment, citrate increased npd by - mv, which suggests that citrate increased clsecretion. in contrast, the other dicarboxylic acids and calcium chelators exhibited little response. conclusion the combination of these responses suggests that citrate exerts complex effects on airway ion transport, most likely dual effects of decreased na + absorption and increased clsecretion. aim to assess the validity of the international physical activity questionnaire (ipaq) in cf adults by comparing energy expenditure measured by the ipaq versus the accelerometer. methods with ethics approval, suitable successive adult patients with cf attending the alfred cf outpatient clinic were recruited. all participants wore an accelerometer (actigraph gt m) around the waist for days of awake time, at the end of which, they completed the ipaq. criterion validity of the ipaq was assessed by comparing the ipaq weekly energy expenditure (ee) in kilocalories (kcal) with weekly ee (kcal) from the accelerometer using spearman correlations and bland-altman procedures. results thirty participants ( % females) completed the assessment: mean (sd); age = ( ) years, fev %predicted = ( ) the median (range) ee: ipaq = ( , ) kcal, gt m = ( , ) kcal. spearman correlations of fev %predicted with ee were gt m ee r = . , p < . ; ipaq ee r = . , p > . . correlation of the ipaq ee with accelerometer ee was moderate (r = . , p = . ). there was a trend towards higher ee measured by the ipaq than measured by the accelerometer (wilcoxon signed ranks test: z = − . , p = . ). conclusion the ipaq underestimates physical activity for patients with lower energy expenditure activities and overestimates for those with higher energy expenditure activities in adults with cf. the ipaq would be a useful screening tool for exercise prescription and monitoring of physical activity longitudinally, but more quantifi able methods for assessment such as the accelerometer should be used in research. confl ict of interest none. infectious endometritis associated with pseudomonas aeruginosa (pa) is an important equine disease resulting in reduced fertility and decreased foal drop. previous typing studies of equine pa report clonal heterogeneity, suggestive of sporadic acquisition, and small clusters of indistinguishable strains. aim we performed molecular typing of a large sample of genital pa isolates from horses in s-e qld. methods thoroughbred genital tract pa isolates submitted to uq vet diagnostic lab during - (screening or infection suspected) were studied. eric-pcr fi ngerprint analysis was performed. isolates producing indistinguishable fi ngerprints were allocated to the same eric-pcr type. mlst was performed on a subset of isolates. results overall, genital (clitoral or uterine) swabs from mares and urethral fossa swabs from stallions located on stud farms were processed. pa was identifi ed in genital cultures from of the ( . %) mares but from none of the stallions. six clusters involving ≥ mares were detected. cluster-a was observed amongst isolates collected from ( %) mares from studs and each year. cluster-b isolates were present in mares from studs during - . clusters c-to-f each contained isolates from mares from or studs. conclusions overall, % of mares harbouring pa had clonally related strains. however, we found no evidence of horizontal transmission between stallions. these data raise the possibility of transmission via environmental or other sources. alternatively, specifi c strains may have trophism for the reproductive tract of horses. the fi nding of a dominant strain amongst thoroughbred mares in a geographic region has interesting parallels with recent evidence of the spread of highly prevalent clonal strains in cystic fi brosis clinics. aim to investigate the prevalence and impact of incontinence in adult men with cystic fi brosis (cf) as compared with age-and sex matched control subjects. methods men with cf were recruited through outpatient clinics and control subjects through advertisements to complete standardized questionnaires relating to respiratory symptoms, bladder and bowel function, mood and physical activity levels. demographic data were collected from medical records for the cf group. results seventy-four men with cf participated (mean (sd) age . ( . ) years). forty-nine men volunteered as controls ( . ( ) years), and were well matched in terms of physical activity levels. / ( %) in the cf group and / ( %) in the control group had reported episodes of urine leakage. in the men with cf, there was no difference in lung function between men with episodes of leak and those with no history of leak (fev % predicted ( )% vs. ( )%, p = . ). anxiety levels were higher in men from both groups with episodes of leak compared to those with no history of leak (hospital anxiety and depression anxiety score . ( . ) vs. . ( . ), p < . ). depression scores were also higher in men with episodes of leak compared to those with no history of leak ( . ( . ) vs. . ( . ), p < . ). conclusions urinary incontinence in men with cf is not associated with disease severity, as measured by lung function. anxiety and depression levels were higher in men with leakage of urine. confl ict of interest no. aim to investigate the bone mineral status of children and adolescents with cf and to explore the relationship between bone mineral density (bmd) and anthropometric and clinical parameters including height, body mass index (bmi), lung function tests and vitamin d levels ( -hydroxyvitamin d) in the cf centre at starship children's hospital, new zealand. methods bmd of the lumbar spine was assessed by dual x-ray absortiometry between january and december . the results of subjects with cf ( males) with a mean age of . years (range - . years) were collected. anthropometric data (height, bmi), forced expiratory volume in second as percent predicted (%fev ) and vitamin levels were assessed and related to bmd. results bmd in our subjects was low in . % and very low in . % when adjusted for age, sex and height (difference in bmd g/cm in the lumbar spine l -l ). there was a strong positive relationship between the lumbar areal bmd (abmd) and bmi z scores (p < . ), abmd and % fev z scores (p < . ), and abmd z scores and vitamin d levels (p < . ). conclusions bmd was normal in the younger and well-nourished subjects with normal or mild reduction of fev . low bmd appeared to evolve during adolescence with decreasing bmi and reduction in lung function. this will lead to ongoing bone disease in early adulthood. it is a further indication to maintain optimal nutritional status and maximize lung health. malnutrition in cf is associated with poorer pulmonary function and is an independent risk factor of survival. aim to compare the nutritional status of the adults attending an adult cf centre in with . method retrospective audit of patients ( excluded, incomplete data) including demographics, nutritional status, pancreatic enzyme replacement therapy (pert) usage, glucose tolerance and dietetic review. results the mean age of the clinic population increased from . to . years. mean (sd) bmi increased from ( . ± . kg/m ) to ( . ± . ) (p = . ). in , % of the clinic population was taking pert with a mean dose of ± iu lipase/kg/day. the proportion of patients with abnormal glucose tolerance has increased from % to % (p = . ). oral supplement use has increased from % to %, yet enteral feeding remained stable ( % − , % − ). this occurred during period of increased annual dietetic review of the patients attending the clinic from % in to % in (p = . ). discussion over a -year period, an improvement in mean bmi refl ects improvement in nutritional status. prevalence of abnormal glucose tolerance has increased; this is likely due to commencing a screening program ( ). use of oral supplements has increased and is higher than identifi ed in the recent daa survey of nutrition practices of cf dietitians ( %). annual review by the cf dietitian has increased despite a twofold increase in the cf population may be attributable to a stable and experienced workforce. current service provision of . a abbott , e cheung , l morgan aim to characterize the microbial colonization of a group of stable adults with non-cf bronchiectasis using an extended culture protocol. methods sputum was collected over an -month period from clinically stable patients. standard semi-quantitative bacterial culture was extended to days with the addition of fungal and mycobacterial culture as routine. results specimens of spontaneously expectorated sputum were collected from patients; mean age years ( - years); mean (sd) fev / fvc ratio % ( %); / never smokers; / on inhaled or oral corticosteroids. the bacteria identifi ed were p. aeruginosa ( % of specimens), h. infl uenzae ( %), h. parainfl uenzae ( %), acinetobacter baumanii ( %), enterobacteriaceae ( %). commensals only were identifi ed in % of specimens. fungi included candida species ( %), aspergillus fumigatus ( %) and penicillium species ( %). non-tuberculous mycobacteria (ntmb) were grown in % of specimens: m. gordonae ( %), m. intracellulare ( %) and m. lentifl avum ( %). the ntm identifi ed were all considered non-pathogenic. only the mycobacteria were identifi ed after day . conclusion microorganisms with potential pathogenicity are frequently identifi ed in adult patients with non-cystic fi brosis bronchiectasis who are not experiencing an acute exacerbation. all these organisms were identifi ed using a standard short culture protocol. the extended regimen, which was costly, did not identify any unusual or unexpected pathogens. it was rare for patients to be colonized with fungi. this study suggests there is limited value in requesting extended culture for bacterial pathogens, including looking for fungi or nmtb in this stable patient group as this adds little to the empiric antibiotic choice for infective exacerbations. confl ict of interest none. s stelzer-braid , , h alsubie , a neilsen , h johal , a steller , er tovey , k mckay , p van asperen , wd rawlinson , introduction respiratory infections are of fundamental importance in determining the morbidity and mortality associated with cystic fi brosis (cf) as such infections can lead to progressive and fatal obstructive lung disease. using polymerase chain reaction (pcr) to detect such infections has advantages over previous studies that used relatively insensitive traditional detection methods and could have underestimated viral prevalence. methods viral and bacterial multiplex pcrs were developed for detection of respiratory pathogens important for children with cf. nasal brush samples were collected from cf patients who were symptomatic or asymptomatic for acute respiratory illness (n = ). sputum and exhaled bioaerosols via a novel mask sampler were collected from a subset (n = ). results as expected, almost all ( %) sputum samples were positive for bacteria. detection of bacteria in the upper respiratory tract was lower ( . %). data from nasal samples indicated strong association of viral pathogen presence, particularly rhinovirus, with exacerbation of disease. results also showed good evidence for rhinovirus infection in the lower respiratory tract. the novel mask sampler is promising as a non-invasive sampling tool. conclusions our results demonstrate the importance of pathogens in exacerbations. early detection and understanding the development of bacterial and viral infections in cf patients is important in clinical decision-making as more and better antiviral and antibiotic agents become available. aim to determine the factors affecting microbiological yield from bronchoalveolar lavage (bal) in patients with suspected pulmonary infection and haematological malignancy or following stem cell transplantation at a tertiary bone marrow transplant centre. methods a retrospective -month audit of patients with pulmonary infi ltrates or febrile neutropenia with haematological malignancy or post-stem cell transplant who underwent bal for microbiological diagnosis. data were obtained on microbiological yield, radiographic appearances, current antimicrobial therapy, the presence and duration of neutropenia and complication rate. of the bal procedures performed, a clinically signifi cant microbiological result was obtained in % of cases ( / ). of these positive results, % ( / ) were exclusively viral pathogens, % ( / ) were fungal, % ( / ) were bacterial and polymicrobial infection was observed in % ( / ) of cases. a high proportion of patients had commenced anti-microbial treatment empirically, with % ( / ) receiving broad spectrum antibacterial treatment and % ( / ) receiving treatment doses of antifungal agents prior to bronchoscopy. in % ( / ), the results of the bal changed the patients therapy. the presence and duration of neutropenia or radiological appearances were not reliable discriminators of specifi c infective aetiologies. complication rates were low and included fevers in % ( / ), hypoxia % ( / ), small volume haemoptysis in % ( / ), atrial fi brillation in % ( / ) and pneumothorax in % ( / ). conclusion whilst bal remains a safe and important tool in establishing a microbiological diagnosis in immunosuppressed patients with pulmonary infi ltrates, a clinically signifi cant yield and changes to patient treatment occur in the minority of cases. clinicians should have a high degree of suspicion of viral infective aetiology when treating this population of patients. aim to examine the outcomes and complications of intercostal catheter (icc) treatment of pneumothoraces (primary (pp) and secondary (sp)) and effusions (malignant (me) and parapneumonic (pe)). methods retrospective review of all iccs in admitted patients in a respiratory unit over months. data collected included type of pneumothorax or effusion, icc type, insertion details, complications (major and minor) and outcome (success defi ned as resolution of pneumothorax or effusion with single tube insertion). results patients required icc treatment. forty-six iccs were used in patients with pneumothorax: pp ; sp ; iatrogenic ; hydropneumothorax . complication rate was % ( % major) and was signifi cantly less in pp ( %) compared with sp ( %), p < . , chi-square. success rate for pneumothorax icc drainage was % (signifi cantly higher for pp ( %) compared with sp ( %), p < . ). fifty-eight iccs were used in patients with pleural effusions: me , pe , other . complication rate was % ( % major) and was signifi cantly higher in me ( %) compared with pe ( %), p < . . success rate for effusion icc drainage was % (signifi cantly less in me ( %) compared with pe ( %), p < . ). small bore iccs (gauge < fr) were used for % of pneumothoraces and % of effusions. tube size did not signifi cantly infl uence complication or success rate for either pneumothoraces or effusions. conclusions compared with pp, icc treatment of sp was less successful and more likely to be associated with complications. similarly, compared with pe, intervention for me with icc was less successful and had a higher complication rate. we conclude that icc intervention is most successful for pp and pe, and speculate that sp and me should have early surgical intervention. introduction spontaneous pneumothorax is a common condition. current management guidelines recommend large pneumothoraces are managed by primary intercostal catheter insertion. we report a single centre's experience in the management of large spontaneous pneumothorax. methods retrospective audit of cases of spontaneous pneumothoraces managed at the prince charles hospital between january and december . patient demographics, co-morbidities, presenting symptoms, examination fi ndings, radiology, management and complications were reviewed. results forty-two patients ( male, female) experienced episodes of spontaneous pneumothorax. chest pain and dyspnoea were the most commonly reported symptoms ( ) %. there were forty-two ( %) episodes of large pneumothorax (≥ % of hemithorax). management of large pneumothoraces consisted of: observation, ( ) seldinger icc ( ) and large bore icc ( ). complications occurred in three patients with seldinger icc ( vasovagal, hydro-pneumothorax) compared to none with large bore icc. outcomes were similar for patients managed by observation compared to icc insertion. all recurrent cases ( %) were referred for consideration of surgical pleurodesis. conclusion patients with large pneumothorax managed by observation recovered similarly to those treated with icc, suggesting a higher threshold for icc insertion should be considered in the future. grant support nil. aim a pilot study of an instrument of pleural ultrasound training in thoracic physicians after a pleural ultrasound course. the instrument was tested for inter-observer agreement and also its ability to be used in a patient compared to a dedicated manikin. methods all chest physicians ( ) were novices in ultrasound and underwent a dedicated -day training course in pleural ultrasound at the australian institute of ultrasound. they were assessed months later by radiologists and one senior ultrasonographer using a specially designed pleural ultrasound training assessment tool (usgt-sat) on both a subject with pleural effusion and a dedicated ultrasound manikin. the mean scores, out of a maximum of , obtained by the each of the participants for the manikin were . , . , . and . , respectively, while the scores for the patient was . , . , . and . , respectively. the mean scores of the participants as a group for manikin were ± . and for the patient as . ± . . there was general agreement between the examiners with mean combined participant scores of . , . and . in the manikin, respectively, and mean score of . , . and . in the patient. conclusions this pilot study shows ranges of scores for design of future validation studies of the usgt-sat. test performance by the chest physicians after a short course in pleural ultrasound was generally good and results for the use of the manikin as an alternative to patients in pleural ultrasound training are encouraging. further studies with larger sample size are required. supported by nil. nomination nil. confl ict of interest no. since the fi rst commercial availability in , fl exible bronchoscopy has evolved from a simple 'look see' procedure to a more complex multifaceted one. today, fl exible bronchoscopy is a tool used for diagnostic procedures, surveillance, delivery of therapy and clinical trials. increasingly, it involves utilizing expensive purpose built equipment in complex diagnostic procedures. this evolution requires a specifi c knowledge base and skill set to safely perform the procedure and care for the equipment. this now mandates additional training by nursing and medical staff to develop and maintain the required skills. medical staff now rely on their nurses to assist in the full range of procedures. thus, the nurses must keep abreast of modern trends and techniques. the modern bronchoscopy suites team is an integrated one, with specifi c roles, defi ned to each member. the procedures performed will refl ect local needs and expertise. just as bronchoscopy has evolved into the speciality of interventional pulmonology, so must bronchoscopy suite nursing be accepted as a specialized area of nursing with a credentialed 'special interest group' to promote, educate and develop the subject as more therapeutic and diagnostic procedures evolve. this will allow nurses involved in bronchoscopy to be respected, recognized and accepted for their unique knowledge and abilities. confl ict of interest nil. background transthoracic pneumostomy (tp) is a novel treatment for patients with severe emphysema that aims to defl ate the lung and improve function. aim to assess the effect of unilateral tp on the volume of each lung and mechanical properties of the lungs. methods subjects were recruited for a multicentre trial of tp (see actrn ). in parallel with the main protocol, we measured ( ) in the six subjects recruited, compared to plethysmography, lung volume was overestimated by cxr (mean difference + . %, range − . to + . ) and underestimated but more closely correlated by ct (mean difference − . %, range − . to − . ). based on ct, the volume of the treated lung decreased in all patients after tp (mean − . %, range − . to − . ) whilst that of the untreated lung did not change (mean − . %, range − . to + . ). in patients with available data, tp reduced dynamic hyperinfl ation during exercise (mean − ml, − . % of ic, range + . % to − . %). lung mechanics were performed in patients. low lung elastic recoil prior to tp and an increase in elastic recoil after tp were associated with greater reductions in lung volume and greater improvements in exercise tolerance. conclusions supine chest ct provided reasonably accurate estimates of plethysmographic lung volume. unilateral tp defl ated the lung and there was no evidence of signifi cant compensatory hyperinfl ation of the contralateral lung. tp also reduced dynamic hyperinfl ation. measurement of lung elastic recoil may help select patients who are likely to benefi t from tp. support and confl ict of interest nil. methods we performed a retrospective chart review of all adult patients who had an icc over a -month period within a tertiary hospital respiratory service. we noted patient demographics, details surrounding chest drain insertion including image guidance and subsequent inpatient events. results over a -month period, there were small-bore icc insertions, of which were image-guided. mean patient age was years, males comprised / . forty drains were inserted for pneumothoraces, for malignant effusions, for parapneumonic effusions, for transudates and for undiagnosed exudative effusions. mean duration of drainage was . days. there were no life-threatening complications. three of the chest drains fell out and became blocked. six pneumothoraces were noted, all following insertion without direct image guidance; none required further intervention. local infection occurred in patient. insertion details were not documented in patients. conclusion insertion of small-bore iccs via the seldinger technique appears to be a safe method of draining pneumothoraces and pleural effusions. image guidance may reduce complication rate of this procedure. documentation of drain insertions could be improved. confl ict of interest nil. rationale pleural effusions are frequently encountered in clinical practice, and often require aspiration for diagnostic and/or therapeutic purposes. use of radiological guidance varies, despite current guidelines recommending routine use of ultrasound. furthermore, concerns exist regarding the downskilling of thoracic medicine trainees due to the increased use of interventional radiology. as a precursor to developing a procedural pleural ultrasound service, we performed a retrospective case review of our current practice. methods patients who had pleural fl uid sent to pathology between january and december were identifi ed on an existing database. patient records were reviewed and details regarding the drainage procedure and outcomes were recorded. information on patient location, method of procedure and performing clinician were also collected. results to date, pleural fl uid aspirations in patients have been identifi ed. overall, % of aspirations were carried out on the ward and % in the radiology department. two procedures occurred in the endoscopy suite on outpatients, and one in the emergency department. fifty percent of procedures were performed using an intravenous cannula for drainage and % utilized a pigtail catheter. all procedures occurring in the radiology department were performed under ultrasound guidance by a radiologist or radiology registrar. of the remaining procedures, % were performed by medical registrars and % were performed with ultrasound marking. six complications occurred following procedures: pneumothoraces, vasovagal and tube blockage. there were signifi cantly more pneumothoraces in patients who did not have an ultrasound marking ( of without marking, of with marking, p = . ). none of the complications required further intervention. conclusion these preliminary data suggest ultrasound marking signifi cantly reduces pneumothorax incidence, supporting the establishment of a pleural ultrasound service. this is likely to have the added benefi t of improved training for thoracic medicine trainees. aim to investigate differences between semi-recumbent and supine posture in terms of cough rate, degree of oxygen desaturation, oxygen supplementation, increase in pulse rate and sedative use during the initial phase of bronchoscopy. methods consecutive patients (n = ) undergoing diagnostic bronchoscopy at an endoscopy unit were recruited for this observational cohort study. the posture was determined by the bronchoscopist's usual practice. patient age, gender, % predicted fev and fvc, indication, pulse and oxygen saturation were recorded. the initial phase was defi ned as the time from bronchoscopy insertion to visualization plus lignocaine instillation of both distal main bronchi. cough rate, peak pulse, nadir oxygen saturation (spo ), range of oxygen supplementation and sedation use during the initial phase were recorded. a post-procedure questionnaire was administered to the patient and the attending nurse. results patients had bronchoscopy in the semi-recumbent posture and in the supine posture. three of bronchoscopists performed in both postures. there were no signifi cant differences in age, gender, smoking status and spirometry between the two groups. the semi-recumbent postures resulted in signifi cantly less cough rate (mean (sd) . ( . ) vs. . ( . ) coughs/min, p = . ) and less fentanyl use ( ( ) vs. ( ) mcg, p = . ) in the initial phase. there were no signifi cant differences in the nadir spo , fall in spo , oxygen supplementation or increase in pulse rate between the two groups. nurse perception of patient discomfort was lower in the semirecumbent position ( ( ) vs. ( ) mm on mm visual analogue scale, p = . ), and there was a trend towards less patient-perceived cough during the procedure in the semi-recumbent group ( ( ) introduction pulmonary infi ltrates in immunocompromised patients with haematological malignancy have a diverse aetiology and are a major source of morbidity. a specifi c diagnosis and targeted therapy may optimize outcomes and reduce the cost of treatment. the diagnostic value of fi breoptic bronchoscopy (fob) and the infl uence of timing of the procedure are unclear. aim to determine the yield of fob, its impact on antibiotic therapy and the infl uence of early vs late timing in this patient population. methods we conducted a retrospective review of immunosuppressed patients with underlying haematological malignancy and new pulmonary infi ltrates who underwent fob over a -month period. the outcomes of early (eb, ≤ days from initial respiratory consultation) and late (lb, ≥ days) fob were compared using fisher's exact test. results thirty-eight fobs, including bronchial or transbronchial biopsies, were performed in patients (males ). there were patients who received eb and who received lb. a specifi c diagnosis was obtained from procedures ( %), including infections ( in eb vs. in lb, p = . ) and non-infective diagnoses ( eb vs. lb, p = . ) based on histology. fob fi ndings from procedures ( %) ( eb vs. lb, p = . ) resulted in modifi cation of antibiotic therapy. there were no procedure-related severe complications. conclusions fob is a useful diagnostic procedure which infl uences diagnostic and therapeutic decisions in this patient group. although early procedures tended to be more likely to change antibiotic therapy than late procedures, the difference was not signifi cant. confl ict of interest none. capsule endoscopy is increasingly performed in gastroenterology to investigate possible small intestinal bleeding. the capsule endoscope is swallowed and then takes photographs every seconds for hours during its transit through the gastrointestinal tract. the images are downloaded by a radio link and the capsule is then passed normally and disposed of. in the present case, the capsule endoscope was inhaled and lodged in the bronchus intermedius. this was only recognized when the images from the capsule download were examined. removal of the capsule was effected with a fi breoptic bronchoscope using an ercp balloon and roth basket. this is believed the only capsule bronchoscopy so far reported. capsule endoscopes are large ( mm × mm diameter) and smooth. this case report shows the images from the capsule endoscope and describes the methods necessary to remove this unusual foreign body from the lung. support nil. background bronchoscopy with endobronchial biopsy (eb) is now an integral component of the research evaluation of airway diseases. there are no published safety data for eb in adult non-cf bronchiectasis. methods a subgroup of subjects enrolled in the bronchiectasis and low dose erythromycin study (bless) a randomized controlled trial of long-term prophylactic erythromycin (anzctrn ) underwent bronchoscopy with bronchoalveolar lavage (bal) and eb performed by a single operator. results ninety-nine bronchoscopies were performed (bal alone in ) in subjects. of procedures with eb, ( . %) were associated with very signifi cant bleeding (> ml either at time of eb or several days post-procedure) and a further ( . %) with immediate moderate bleeding ( - ml). one subject had a history of prior signifi cant haemoptysis. in the four subjects with very signifi cant bleeding, immediate bleeding of > ml occurred in subjects, ml in one subject and ml in one. immediate bleeding was controlled uneventfully. three of the subjects subsequently developed signifi cant haemoptysis (> ml) to days post-bronchoscopy without intervening haemoptysis, with one subject developing massive haemoptysis (> ml) on day post-bronchoscopy. further research ebs were ceased. in one of the subjects with 'delayed rebleeding', repeat bronchoscopy confi rmed the biopsied lobe as the bleeding site. haemoptysis settled in all subjects within hours with simple conservative measures. conclusions in contrast to the experience in asthma and copd, research eb in adults with non-cf bronchiectasis is associated with a signifi cant risk of bleeding, of potentially life-threatening magnitude in . % of cases. of particular concern was the observation of sudden onset delayed rebleeding developing up to days post-eb in spite of early local control. histopathological evaluation will clarify the potential contributions of airway wall vascularity and infl ammation to these events. malignant mesothelioma (mm) is an aggressive cancer which is often associated with exposure to asbestos and sv . this disease has a high latency period and a low survival rate. therefore, new strategies for therapeutic intervention must be developed. recent studies have shown that developmental pathways including the hedgehog (hh) pathway are associated with various types of cancers. the aberrant activation of key hedgehog pathway proteins has been shown to contribute to cancer progression. however, the role of this pathway in mm has yet to be explored. we hypothesize that aberrant activation of the hh pathway is a contributing factor for the development of mm. the mrna expression of hh pathway genes; sonic hedgehog (shh), patched - (ptch- ), smoothened (smo) and gli- were examined in mm cell lines and tumour tissues by rt-pcr and qrt-pcr. hh pathway proteins and mrna expression and distribution were then observed in the tumours by immunochistochemistry and in situ hybridization. we used real-time superarrays to examine the change in expression of a panel of key hh pathway genes by activating and inhibiting the pathway. we showed that the key hh pathway genes are expressed in both the cell lines and tissue samples. upon stimulation with the ligand shh, there was an increase in expression of indian hedgehog (ihh) and shh in most of the mouse and human cell lines that we looked at. interestingly, for the transcription factor gli- , there was a significant decrease in both mouse and human cell lines. inhibiting this pathway increased the expression of ptch in the mouse and human cell lines. the expression and up-regulation of key hh pathway components in mm at baseline and following stimulation suggests a role for the pathway in mm. methods incident cases were obtained from the australian and wa mesothelioma and cancer registries and death registries. exposure was calculated using measures of dustiness in the industry and the town for the period of employment or residence of each case. latency (time from fi rst exposure to diagnosis) by sex, age, smoking status, exposure variables and worker or resident status was estimated. multivariate linear regression modelling examined the determinants of latency. results the mean latency periods of . (sd = . ) years for lc and . (sd = . ) years for mm have increased linearly. increased duration of exposure was associated with reduced latency for mm after adjustment for age at fi rst exposure and age at diagnosis but not signifi cantly for lc. age at diagnosis was strongly associated with latency length for both lc and mm (p < . ). smoking, sex, cumulative exposure (log f/ml-year) and status at wittenoom were not related to latency. latency for lc with increasing age at fi rst exposure declined faster than for mm. conclusions age at diagnosis is associated with reduced shorter latency of mm and lc. duration of exposure is associated with shorter latency of mm. supported by nhmrc australia. confl ict of interest no. aim to assess overall survival of patients following resection for stage nsclc at a centre that has substantially greater resection rates than the nsw average. methods a retrospective audit of those patients who underwent lung resection for stage nsclc at nepean hospital between january and february . results patients ( m: f), mean age (range - ) underwent resection. there were pneumonectomies, bilobectomies and segmentectomies, one involving chest wall resection. the remaining procedures were lobectomies. there was one perioperative death from respiratory failure. actuarial overall survival at months was %, at months, % and at years %. survival was not infl uenced by histology or age. conclusion in our institution, we have an agreed aggressive approach to resection of stage nsclc and our resection rate is %. this pro-surgical policy is associated with good perioperative and long-term overall survival. confl ict of interest no. introduction malignant pleural effusions (mpes) are common, although their management varies widely. providing ambulatory care to minimize hospitalization is a key goal for patients with mpes. indwelling pleural catheters (ipcs) are a new treatment strategy that allows outpatient fl uid drainage. we hypothesized that mpe patients managed with ipcs require fewer hospital admissions. methods a prospective, multicentre, non-randomized study involving all three major respiratory centres in western australia. patients diagnosed to have mpes were prospectively followed, and admissions were recorded. in the absence of accepted guidelines for ipc use, the choice of treatments (thoracentesis, ipc, pleurodesis) was decided by clinicians in-charge. all complications were recorded. bacterial cultures of pleural fl uid were performed monthly for patients with ipcs. hm gallagher , ee duhig , ia yang , rv bowman , be clark , hm marshall , km fong aim to determine the concordance of histological subtyping of nsclc in diagnostic samples to the gold-standard lung resection specimens. methods we have so far evaluated consecutive subjects who underwent curative surgery for primary nsclc at the prince charles hospital between the years and . many of these had workup at other institutions. one hundred forty-seven had queensland health electronic record of positive preoperative diagnostic sampling. we correlated the fi nal nsclc who histological subtype with the subtypes diagnosed by samples prior to surgery including sputum, fi beroptic bronchoscopy (fob) and trans-thoracic needle aspiration (ttna). the resection subtype was set as the reference standard, and concordance was compared. results of the cases of resected nsclc, had malignancy on diagnostic sampling pre-resection, as shown in the results patients were included: median age years (range - ); % male; % living in major cities versus % in regional areas; % rightsided mpm; % epithelial subtype. median time from asbestos exposure to diagnosis was years (range - ). median time from fi rst symptoms or investigations to diagnosis was weeks (range - ). all patients had at least one chest x-ray and ct scan and % had pet scan. a variety of procedures led to the diagnosis: % thoracoscopy, % thoracotomy, % radiology-guided, % chest wall biopsy and % medical pleuroscopy, with % having had cytology alone. median number of diagnostic immunohistochemical stains used was (range - ), with calretinin ( %) the most commonly used mesothelial marker and carcinoembryonic antigen (cea; %) the most common carcinoma marker. median os for the cohort was . months ( % ci: . - . ), with no statistical difference in os between major city and regional patients ( vs. . months, respectively, p = . ). conclusions mpm appeared to affect mainly the elderly, and thoracoscopy was the most common diagnostic procedure. os did not differ between australian major city and regional patients and was comparable to the largest phase iii trial in mpm. aw musk , , p aboagye-scarfo , a reid , a miller, s ruwanpura, l mcleod, p bardin, n watkins, bj jenkins rationale lung cancer is the leading cause of cancer death worldwide. it is well established that cigarette smoking is linked to emphysema and lung cancer, and smokers with emphysema are at an increased risk of developing lung cancer. notably, recent epidemiological studies have indicated that emphysema can predispose to lung cancer irrespective of pack-year smoking history. although infl ammation has been proposed as a common mechanism linking these two diametrically opposed diseases, the conceptual inter-relationship between infl ammation, emphysema and lung cancer has been poorly investigated because existing experimentally induced and genetically modifi ed animal models for lung cancer occur in the absence of emphysema. method we have utilized a newly identifi ed mouse model (gp f/f ) of spontaneous lung infl ammation and emphysema in two well-established lung cancer models. the gp f/f mouse is characterized by deregulated cytokine signalling via gp , the critical co-receptor for the interleukin (il)- cytokine family, leading to hyper-activation of stat , a potent pro-infl ammatory and oncogenic latent transcription factor. in separate studies, we exposed gp f/f mice to a cigarette-derived carcinogen (nnk), and crossed them with the genetically susceptible kras(g d) strain of mice. results in both nnk-and kras(g d)-induced lung cancer models, the lungs of gp f/f mice were highly predisposed to hyperplasia and tumour formation. increased levels of cellular proliferation were observed in hyperplastic and tumour lesions, as well as surrounding areas, of these mice. these observations were verifi ed at the molecular level by gene expression profi ling of tumour-bearing lung tissue. conclusions these studies provide unique insights into the importance of interactions between the gp signalling axis and factors that predispose to lung tumourigenesis in emphysema. support nhmrc. aim to assess the preparedness of hospitals with respect to protecting health-care workers (hcws) during a pandemic. methods a self-administered questionnaire was performed between november and january , and a scoring system was developed to provide a quantifi able measure of preparedness. results a total of hospitals in nsw, australia, were approached -six regional hospitals (rhs) and six tertiary referral centres (trcs). the study was extended to assess three hospitals in england, allowing a limited comparison between the hospitals in australia that had faced the initial wave of the h n ('swine fl u') pandemic and the hospitals in the uk that had more time to prepare for the outbreak. response rates were % from the trcs, % from the rhs and % from the english hospitals. the overall preparedness scores were relatively high, with a median total score (adjusted) of . out of . the demographic that scored the highest total was tertiary referral centres in sydney. all english hospitals scored below the median. however, the range of scores across hospitals was quite narrow ( . - . adjusted). scores were generally high for the areas of preparedness, infection control, education and training. scores for vaccination were more variable. the category that consistently demonstrated the lowest scores was that of psychosocial welfare and assistance, despite this found in previous research to be an integral part of that which hcws have identifi ed as important. conclusions given their integral role in pandemic response, protecting hcws must be a priority as part of any pandemic preparedness plan. this goes beyond protection from infection, extending into aspects of physical and psychological wellbeing. identifying these issues and addressing them is the key to maximizing staff support and morale, and minimizing staff absenteeism at such a crucial time. aim to describe the relationship of respiratory and refl ux symptoms within the general population and relate this to the possible confounding factors of body mass index (bmi) and obstructive sleep apnoea (osa). methods data from a cross-sectional health survey, performed in bussleton, west australia in - , were used to examine the relative effects of bmi and osa on the relationship between respiratory and refl ux symptoms. questionnaire data included information on asthma, cough, wheeze, dyspnoea and gord symptoms. gord symptoms were categorized as never, monthly or less often and weekly or more often. bmi, risk of osa defi ned according to the berlin questionnaire, spirometry and airway hyperresponsiveness to methacholine were also recorded. logistic regression models obtained odds ratios for the associations between each gord symptoms, various respiratory symptoms, bmi and osa. results average age was years and recent wheeze was reported in % and cough and phlegm in %. twelve percent were current smokers. ahr was present in % and osa in %. gord symptoms occured in % and frequent symptoms (weekly or more often) were present in - %. there were strong positive associations between gord symptoms and cough/phlegm, breathlessness, chest tightness and wheeze in the last months. odds ratios increased with increasing frequency of refl ux p ≤ . . there was no effect of obesity or osa on the relationship between respiratory and gord. conclusion cough and phlegm, breathlessness, chest tightness and wheeze (ever or recent) are all strongly associated with symptoms of gord. this relationship is amplifi ed with increasing frequency of gord symptoms indicating a dose-response relationship between refl ux and respiratory symptoms. obesity and osa do not affect the association between gord and respiratory symptoms. introduction diesel exhaust particles (dep) make up the bulk of particulate matter in urban areas. high ambient levels of particulate matter are associated with increased hospitalization due to respiratory disease. we aimed to determine if exposure to dep exacerbates responses to acute viral infection. methods adult female balb/c mice were inoculated with μg dep or control . days after infection with . plaque forming units (pfu) of infl uenza a/mem (or control). six hours after dep inoculation, lung volume (tgv) and lung mechanics were measured by plethysmography and the forced oscillation technique, respectively. bronchoalveolar lavage fl uid was collected to assess cellular infl ammation and cytokine levels. results viral titre was signifi cantly higher in infl uenza-infected mice exposed to dep compared to those exposed to infl uenza alone (p = . ). both dep (p = . ) and infl uenza infection (p < . ) alone signifi cantly increased cellular infl ammation; however, there was no difference between mice exposed to both dep and infl uenza compared to those exposed to infl uenza alone (p = . ). a similar pattern was found in levels of cytokines in the bronchoalveolar lavage (tnf-α, mcp- , il- , ifn-γ). specifi c airway resistance, specifi c tissue damping, specifi c tissue elastance and hysteresivity were signifi cantly increased in infl uenza infected mice (p < . in all cases). none of these parameters were infl uenced by dep exposure alone (p > . in all cases) and there was no additive effect of dep on lung function (p > . in all cases) in infl uenza-infected mice. conclusions dep increases viral titre but is not suffi cient to physiologically exacerbate pre-existing respiratory disease caused by infl uenza infection in mice. supported by nhmrc. confl ict of interest no. introduction lack of treatments for post-transplant obliterative bronchiolitis (ob) is mainly due to the poor understanding of its pathogenesis and lack of small airway models. epithelial-mesenchymal transition (emt) may play a central role and could be crucial to developing treatment drugs. we hypothesize that emt induction may be prevented by pharmacologically available compounds. methods primary cultures of small and large airway epithelial cells (saec and laec) were established and emt induced by adding tgfβ ( ng/ml) (n = ). azithromycin ( - μm), mycophenolate ( . - mg/l) and rad ( . - ng/l) were then added and expression of epithelial (zo- , ck- ) and mesenchymal markers (eda-fn, vim) measured via western blot as well as mmp and activity via zymography. results signifi cantly lower increase in mesenchymal markers and lower decrease in epithelial markers, compared to controls was noted for azithromycin and mycophenolate indicating suppression of emt. mmp and activity increase was also signifi cantly suppressed. azithromycin suppressed emt to a greater extent compared to mycophenolate, but was equally effective in both small and large airway epithelia. rad appeared to have no effect. conclusions azithromycin and mycophenolate are both effective in preventing emt and thus have potential for the clinical treatment of ob. supported by abn foundation. confl ict of interest none. journal compilation © asian pacifi c society of respirology tp- g hodge , , s hodge , , c-l liew , , t-cell pro-infl ammatory cytokines are associated with acute lung transplant rejection. we have previously shown compartmentalization of production of these cytokines in bronchial intraepithelial t cells (iet) obtained by bronchial brushings from stable lung transplant patients. during acute rejection episodes, no signifi cant differences in iet cytokines were observed between stable and rejecting patients due to broad cytokine variability between patient groups. to overcome this limitation, we hypothesized that there would be increased graft pro-infl ammatory iet cytokines compared with native lung or trachea during acute rejection. methods cell cultures from stable patients, patients with evidence of acute rejection and bos and healthy controls were stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in graft iet-cell ifnγ and tnfα in the lungs of patients with acute rejection compared with iet cells obtained from the native lung or trachea, but no changes were noted between other patient groups. there was a signifi cant correlation between increased graft iet-cell tnfα compared with trachea and lungs and acute rejection grade. conclusions differential expression of pro-infl ammatory cytokines by iet cells from graft, trachea or native lung distinguishes severity of acute rejection. improved monitoring response using this assay or therapeutic targeting of these pro-infl ammatory cytokines may reduce acute lung transplant rejection. supported by nhmrc. aim to determine the prevalence of reduced carbon monoxide transfer factor (dlco ≤ % predicted) in subjects undergoing pulmonary function testing (pfts) and to determine whether a cause has been identifi ed. methods a clinical audit of all subjects undergoing pfts at royal melbourne hospital from august to august who have a dlco ≤ % in the setting of normal spirometry. medical records and investigations including transthoracic echocardiogram (tte), high-resolution commuted tomography (hrct), ventilation/perfusion (v/q) scans were reviewed to determine whether a cause for the reduced dlco was established. where a cause was not clear, subjects were invited to participate in a telephone interview to evaluate symptoms and to undergo repeat pfts. subjects with a persistently reduced dlco were invited to undergo further investigation with tte, hrct and v/q scan. preliminary results pft results from subjects were reviewed. subjects with fev /fvc < , fev < % predicted and fvc < % predicted were excluded. three hundred seventy subjects ( %) had an isolated reduction in dlco. / ( %) of these subjects underwent tte with / ( %) demonstrating an elevated right ventricular systolic pressure (rvsp). in all cases where there was an elevated rvsp an identifi able cause was found. / ( %) of these subjects subsequently identifi ed as having pulmonary arterial hypertension (pah) and commenced appropriate therapy and / ( %) identifi ed as having pah where treatment was not commenced. there were / ( %) of subjects who appeared not to have undergone a tte. further evaluation of medical records of subjects who had not undergone tte and those with normal tte is continuing. review of subjects hrct, v/q scans and right heart catheterizations is currently proceeding. conclusions preliminary results suggest that a signifi cant proportion of subjects with isolated reduction of dlco on pfts do not undergo tte which is an important investigation in determining the cause for the reduced dlco. when a tte is performed and demonstrates an elevated rvsp, a cause for the elevated rvsp is identifi ed. sponsor actelion pharmaceuticals australia pty ltd. g hodge , , s hodge , , c-l liew , , , pn reynolds , , m holmes , , background t-cell pro-infl ammatory mediators are associated with acute lung transplant rejection. we have previously shown that bos was associated with lack of immunosuppression of t-cell pro-infl ammatory cytokines and increased t-cell granzyme b in peripheral blood. recently, we also showed that nkt-like cells are a major source of pro-infl ammatory cytokines and granzymes in the blood of stable lung transplant patients. we hypothesized that bos may be associated with lack of immunosuppression of these proinfl ammatory mediators in blood nk and nkt-like cells. method granzyme/perforin profi les from stable patients, patients with evidence of bos and healthy controls were determined and blood cultures stimulated and intracellular cytokines determined using multiparameter fl ow cytometry. results there was a signifi cant increase in the percentage of nk cells expressing granzymes and perforin in bos patients compared with stable patients and controls. there was an increase in the percentage of t, nk and nkt-like cells producing ifnγ and tnfα in bos compared with stable patients. there was a signifi cant correlation between increased nk ifnγ and tnfα and fev . conclusions bos is associated with increased peripheral blood nkt-like and nk cell granzymes, perforin and th pro-infl ammatory cytokines. therapeutic targeting of these pro-infl ammatory mediators and monitoring response using this assay may reduce bos. supported by nhmrc. confl ict of interest nil. rationale pulmonary embolism (pe) is the leading cause of maternal mortality in the developed world. consequently accurate diagnosis of pe is critical. this must be tempered by the potential radiation risk of investigations to the mother and foetus. we performed a retrospective case review to determine the incidence of pe in pregnant patients investigated for this condition. demographic information, the diagnostic algorithm utilized and the diagnostic yield of investigations were obtained. method pregnant women who underwent ventilation perfusion (vq) scanning or computed tomography pulmonary angiogram (ctpa) at our institution between january and january were identifi ed by an internal database audit. in addition to demographic data, information about the diagnostic pathway and fi nal diagnosis were collected. in cases where pe was not diagnosed, the medical records were reviewed for any subsequent events up until the date of delivery. results during the fi ve-year period, vq scans and ctpas were performed on pregnant women. the average gestation at investigation was weeks. only one patient had a previous history of venous thrombo-embolism. % underwent doppler ultrasound of the lower limbs prior to vq or ctpa. overall the incidence of pe was %, diagnosed by vq scan. otherwise the vq scans were normal in %, low probability in % and non-diagnostic in % cases. ctpa was non-diagnostic in % of cases. all other ctpa studies demonstrated no emboli. almost % of scans were done after hours ( % vq and % ctpa). no patients without pe were felt to have had the pe missed up to the time of delivery. conclusions the overall incidence of pe in patients being investigated was extremely low at %. during this study period slightly more vq studies were performed than ctpas, with each test having similar diagnostic rates. only % of patients had undergone venous doppler prior to undergoing radiationexposing investigations. nomination nil. introduction anti-ro- antibodies have been associated with idiopathic interstitial pneumonia (iip) in one small series (n = ). we hypothesize that ro- antibodies, just like myositis antibodies, can serve as a marker of undifferentiated connective tissue disease (ctd) with interstitial pneumonia as the primary phenotypic manifestation. the aim of this study was to examine the characteristics of patients with ro- and iip. methods retrospective study identifying patients with iip and ro- positivity, but negative for ctd and/or myositis antibodies, presenting between june and june . data relating to demographics, diagnosis, pulmonary function tests, length of follow-up and outcome were obtained. all hrct images were reviewed by an independent expert radiologist (dm). results / ro- positive subjects fulfi lled criteria ( male, median age ( - ), european, never smoked). / had ro- titers above and in the intermediate ( - ) range. three patients had raynauds phenomenon; there were no other ctd features. / patients had hrct diagnosis of nsip and / organizing pneumonia; / had extensive fi brosis. mean (sd) % predicted baseline fvc ( ), dlco ( ). median length of follow-up was months. all patients were treated and were considered overall stable at last follow-up, one had declined and one died of respiratory failure. conclusion this study confi rms an association between ro- positivity and interstitial pneumonia in the absence of defi ned connective tissue disease, suggesting an autoimmune basis for the interstitial lung disease in this group of patients. a larger cohort is required to determine the true signifi cance of this observation. background community acquired respiratory viral (carv) infections are believed to contribute to morbidity and mortality after lung transplantation, but previous studies have not conclusively established the evidence base in this area. patients and methods a prospective cohort study was performed at a single centre from august to march (n = lung transplant recipients). carv infection (human metapneumovirus (hmpv), respiratory syncytial virus (rsv), infl uenza a (flu a), infl uenza b (flu b), adenovirus and parainfl uenza virus (piv)) was confi rmed using polymerase chain reaction (pcr) of upper (nasopharangeal swab) and/or lower (bronchoalveolar lavage) respiratory tract secretions. carv infection and bos were included as segmented time-dependent covariates in a cox proportional hazards model with death as the outcome variable. results patients ( % of the total cohort) had a total of separate carv episodes: piv, hmpv, rsv, flu a, flu b, and adenovirus. infection with either rsv or hmpv was associated with an increased risk of death (p < . hr . , % confi dence interval, . - . ), and the effect persisted after multivariate analysis. bos was also a risk factor for acquiring hmpv or rsv infection (p = . or . , % confi dence interval, . - . ). conclusions infections with hmpv and rsv, but not other carvs, are associated with an increased likelihood of death. the presence of bos is a risk factor for symptomatic infection with hmpv and rsv. ns harun , k sanders , a stuart , cl steinfort department of respiratory medicine, barwon health, vic., australia, and department of clinical and biomedical sciences, barwon health, vic., australia aims nebulized colistin is used to treat recurrent exacerbations of bronchiectasis due to pseudomonas aeruginosa, a major pathogen regarded as diffi cult to eradicate. this case-control study aimed to establish if long-term colistin use could clear p. aeruginosa from the sputum of adults with non-cystic fi brosis bronchiectasis, and if so, whether colistin could be ceased in these patients. secondary outcomes included effects of colistin on quality of life (qol), symptom control, admission rates, lung function and tolerability. methods ( ) sputum was collected in bronchiectasis patients with p. aeruginosa. clearance rates in those on colistin were compared with a control group not on colistin. ( ) colistin patients cleared of p. aeruginosa ceased treatment. sputum was re-cultured at day and to detect recurrence. ( ) a questionnaire assessing qol, symptom control, and admission rates was performed on patients. outcomes were compared before and after colistin use. long-term colistin side-effects and lung function were also assessed. results ( ) % (n = / ) of colistin patients cleared p. aeruginosa from sputum compared with % (n = / ) in the controls (p = . ). ( ) % (n = / ) of patients ceasing colistin remained free of p. aeruginosa at day . ( ) there was no difference in frequency of breathlessness, sputum production or qol scores between the groups (p > . ). the colistin group had lower fvc ( . vs. . l, p = . ) and higher admission rates ( % vs. %, p = . ). on colistin, % of patients reported reduction in sputum frequency, breathlessness and improvement in qol. fifty percent reported decreased admission rates. there were no colistin side effects. conclusions clearance of p. aeruginosa in sputum is possible. clearance rates were similar in those with more severe bronchiectasis treated with colistin compared with stable patients not on colistin, and may suggest suppression of p. aeruginosa by colistin in this severe group. there are benefi ts of colistin on qol, symptom control and admission rates. continued sputum clearance after colistin cessation is achievable in some patients. nebulized colistin use is well tolerated. nomination janet elder travel award. confl ict of interest no. however, use of such agents is suboptimal in hospital patients. this study aims to determine whether a dedicated multidisciplinary education and reinforcement program improves the use of appropriate vte prophylaxis. methods prior to the education programme, we audited a bed general thoracic medical ward including patients with general medical conditions, lung cancer, chronic obstructive pulmonary disease, lung transplant and cystic fibrosis. our multidisciplinary research team developed and implemented an education program over months, using posters, leafl ets and oral presentations to increase awareness and promote adherence to vte prophylaxis guidelines for health care staff involved in direct patient management. following completion of the program, we reaudited the same bed ward. results prior to the education program, a total of patients (mean age ± ) were identifi ed as appropriate for vte prophylaxis. of these ( %) were on appropriate vte prophylaxis. the post education audit showed out of ( %) patients were on appropriate vte prophylaxis. (p = . ). conclusion an effective multi-faceted educational program can improve delivery of appropriate vte prophylaxis, leading to improved outcomes in hospitalized patients. supported by sanofi aventis. confl ict of interest nil. the anti-rheumatic anti-infl ammatory biological agents in clinical use are abatacept, anakinra, adalimumab, etanercept, infl iximab and rituximab. a variety of pulmonary side-effects have recently been reported for these agents and the purpose of this review is to compile the various reported pulmonary toxicities and their prevalence methods we performed a search of databases ovid medline® and embase of the english literature up to august using the mesh terms of abatacept, anakinra, rituximab, adalimumab, etanercept, infl iximab and respiratory tract disease with limits to include only human studies or case reports. in addition case reports of respiratory adverse effects reported to the australian drug reaction advisory committee (adrac) were obtained in order to identify the most common pulmonary reactions reported with each individual agent. results using the search criteria defi ned above and articles were identifi ed in the ovid medline and embase database respectively. the majority of adrac reports were associated with rituximab (n = ) and infliximab (n = ), followed by adalimumab (n = ) and etanercept (n = ). various pulmonary side-effects including interstitial lung disease associated with anti-infl ammatory agents were identifi ed. discussion from the articles reviewed, details about the duration between onset of treatment and incidence of pulmonary side effects, diagnosis, treatment options and outcome of patients were extracted and are presented here. conclusion this comprehensive systematic review hopes to improve the awareness about the serious and potentially life-threatening pulmonary sideeffects of this group of agents. confl ict of interest no. sj simpson , pd sly , p franklin , e lombardi , c calogero , m palumbo , gl hall , introduction the forced oscillation technique (fot) is effort independent and thus ideal for young children. the area under the reactance curve (ax) has been proposed to amplify clinically relevant signal by taking advantage of any shape change in the reactance (xrs) curve below the resonant frequency. this study aimed to develop reference values for resistance (rrs), xrs and ax in a large healthy population of children, and determine if ax conferred any additional clinical benefi t when examining disease in children born preterm. methods impedance spectra were obtained in healthy children ( male), aged less than years and with height less than cm using a commercial device (i m, chess medical, belgium). ax was calculated in of these children between hz and the resonant frequency. backwards stepwise linear regressions identifi ed the best predictors of ax, and xrs and rrs at hz (xrs , rrs ), and z scores were generated. z scores were calculated for children born preterm, of which received a neonatal diagnosis of bronchopulmonary dysplasia (bpd). chi squared tests examined the difference in proportion of children born preterm (with and without bpd) with abnormal z scores for each fot variable. results all fot variables were predicted by height (p < . ) and sex. mean (sd) z scores for preterm children with and without bpd for rrs ( . ( . ); . ( . )), xrs ( . ( . ); . ( . )) and ax ( . ( . ); . ( . )) were all signifi cantly different (p < . ) from the healthy population. the number of children born preterm with abnormal z scores was not significantly different when comparing ax, rrs and xrs . conclusions while ax is able to detect respiratory disease in preterm children with and without bpd, it is no more sensitive than xrs or rrs. supported by pmh foundation, nhmrc, asthma foundation wa, carivit, ngo 'solidarietà e servizio' viterbo. confl ict of interest no. introduction survivors of preterm birth born with bronchopulmonary dysplasia (bpd) in the pre-surfactant era of neonatal care (classical bpd) have a reduced pulmonary gas transfer capacity. there is, however, little data to describe gas transfer in preterm infants with bpd in the post-surfactant era (new bpd). objective assess gas transfer using carbon monoxide diffusing capacity (dl co ) and its components, pulmonary capillary blood volume (vc) and pulmonary membrane diffusion (d m ), in contemporary survivors of preterm birth. method gas transfer was assessed using single-breath dl co in children aged to years and born < weeks gestation with bpd (pb, n = ) and without bpd (pt, n = ), and in term born controls (tc, n = ). dl co z scores were calculated. d m and vc were determined in pb, pt and tc children. the mean (sd) dl co z score for the pb group was − . ( . ) differing signifi cantly from (p = . ) while the pt and tc groups ( . ( . ) and − . ( . ), respectively) did not (p > . ). d m was lower in the pb group than the pt and tc groups, with no difference between pt and tc groups. differences in d m were not signifi cant after adjusting for lung size. there were no differences in vc between groups. conclusion gas transfer is reduced in survivors of preterm birth with new bpd. the tendency for reduced d m and not vc in children with new bpd suggests that impaired gas transfer may be a result of alterations in the alveolar membrane rather than pulmonary vascular function. background bronchiectasis is common in indigenous populations such as alaska natives, australian aboriginal, and new zealand maori and pacifi ca. as part of an international collaborative interventional study, we sought the participation of maori and pacifi ca families -groups diffi cult to engage in research in the past. aim to engage, enrol and retain children from maori and pacifi ca families from auckland in a -year research study. methods a randomized controlled trial to determine whether azithromycin is superior to placebo in reducing exacerbations seeking to enrol children aged months to years with bronchiectasis. the enrolment procedure was modifi ed to a process deemed more appropriate to these cultures: ( ) request to defer the decision of enrolment until the process had been completed. ( ) a minimum of meetings; initial invitation, discussion in the home with the extended family, invitation to the extended family to participate in the day of enrolment. ( ) appointment of a 'whanau worker' (family worker) to sit with the family and empower them to get all the information they seek prior to enrolment. results of families approached, ( %) children (median age . years, range . - . years) enrolled with % samoan, % tongan, % maori and % mixed maori/pacifi ca heritage. after -year retention was ( %) with exiting the study after month with new non-pulmonary disease, and exiting after year, moving outside study area. conclusions these are high enrolment and retention fi gures reported in this population. we believe that following a prolonged procedure for enrolment, involving the extended family and appointing a worker to sit 'alongside' the family will improve their understanding of a research project and allow them to feel more comfortable about participating. aim bronchiolitis is the most common reason for hospital admission for infants globally ( ) . the use of macrolides for treating bronchiolitis in nonaffl uent settings remains controversial but potentially benefi cial. in our region readmission with lower respiratory illness in young children (particularly indigenous children) remains high. this rct aims to determine if a single dose of azithromycin reduces the morbidity of young children with bronchiolitis. methods double blinded rct. young children ≤ months admitted to royal darwin hospital (rdh) diagnosed with bronchiolitis are eligible. children are given a single dose ( mg/kg) of either azithromycin/placebo. primary outcome is length of stay for respiratory disease. secondary outcomes are duration of oxygen use and readmission for respiratory illness in -month period. respiratory viral infections often lead to exacerbations of chronic respiratory diseases such as asthma and copd though there is no similar data in noncystic fi brosis (cf) bronchiectasis. the objectives of our study were to ( ) determine the point prevalence and identify viruses associated with exacerbations and ( ) evaluate clinical and investigational differences between viral infection positive and negative exacerbations in children with non-cf bronchiectasis. methods a cohort of children (median age years; boys) with non-cf bronchiectasis was prospectively followed for child-months. polymerase chain reaction for respiratory viruses was performed on nasopharyngeal aspirates collected during paediatric pulmonologist defi ned exacerbations. data on clinical, parent cough-specifi c quality of life (pc-qol), systemic markers (crp, il , procalcitonin, amyloid-a, fi brinogen) and lung function parameters were also collected. results respiratory viruses were detected during ( %) exacerbations: picornavirus in episodes [human-rhinovirus (hrv) in , enterovirus in ]; human bocavirus in ; adenovirus, human meta-pneumovirus, infl uenza a, respiratory syncytial virus, parainfl uenza and in two each; coronavirus and parainfl uenza and in one each. viral co-detections occurred in ( %) exacerbations. among genotyped hrv's, more hrv-a's (n = ) were identifi ed than hrv-c's (n = ). children with proven viral infections were more likely to have fever (or . , % ci . - . ), wheeze and/or crackles (or . , % ci . - . ) and raised crp (or . , % ci . - . ) when compared with virus negative exacerbations. there were no other statistically signifi cant differences. conclusions respiratory viruses are commonly found during pulmonary exacerbations in children with non-cf bronchiectasis. hrv-a is the most frequently detected virus. time sequenced cohort studies during stable state, exacerbations and recovery periods are needed to determine the importance of viral infections and their possible interaction with bacteria. supported by anz trustees scholarship. confl ict of interest none. nominations none. to date children enrolled, % rsv+ve. median age . months. fifty percent have had at least one co-morbidity. readmission rate = %. conclusion co-morbidities are high in this population. antibiotics have the potential to help reduce the impact of additional respiratory burden. foundation. introduction foreign body inhalation is a relatively common presentation in young children, especially less than years of age. early recognition remains a critical factor in the treatment of foreign body inhalation in children. inhaled foreign bodies in children are most often organic material, with seeds and peanuts being the most common items. on review of the literature, there are very few case reports of inhaled metal screws. we report two unusual cases of inhaled metal screws that presented to our service. case presentation both cases presented to our emergency department with wheeze, respiratory distress and fever. foreign body inhalation was not considered as a cause for their symptoms until the object was identifi ed on chest x-ray. both foreign bodies were removed successfully but one child required invasive ventilation in our intensive care unit post removal. both children made a full recovery. interestingly, both metal screws came from fl at pack furniture purchased from a well known international home products store. conclusion foreign body inhalation must always be considered as a cause of respiratory distress in a child. with the increase in the number of fl at pack furniture in australian home's, we believe parents must be warned of the potential danger of loose metal screws to young children. supported by none. cough in children is a common symptom. data on causes of chronic cough in young children have previously been published by our units. however, differences in underlying diagnosis by age at presentation have not been assessed. we present the 'time to cessation' of cough in our multicentre rct using a standardized management algorithm in newly referred children with chronic cough (> weeks) from australian centres. methods parents completed validated cough diary and cough specifi c qol (pc-qol) at recruitment and at cessation of cough. the diagnosis made by the treating physician was based on tsanz position statement. results the median (range) age of the children recruited was . years ( . - . ); ( %) were boys. median (iqr) pc-qol post treatment of . ( . , . ) improved signifi cantly (p = . ) from . ( . , . ) at enrolment. the median (iqr) duration of cough at recruitment was weeks ( . , . ) and 'time to cessation' of cough after application of the management algorithm was weeks ( . , . ). there was no signifi cant difference (p = . ) in median (iqr) 'time to cessation' of cough among the three age cohorts: < years (n = , . %) was . weeks ( . , . ); - years (n = , . %) was weeks ( . , . ); and > years (n = , . %) was weeks ( . , . ). there was also no signifi cant difference in the fi nal primary diagnosis among the three age cohorts (p = . ). the most common diagnoses were protracted bacterial bronchitis (n = , %), asthma/reactive airways disease (n = , . %), tracheobronchomalacia (n = , . %) and bronchiectasis (n = , . %). children ( . %) had more than one diagnosis. conclusions the aetiology and 'time to cessation' of chronic cough in children managed in accordance to a standardized pathway were similar among the three age groups. it is likely that our previous fi ndings in very young children are also applicable to older children. supported by nhmrc grant number . confl ict of interest none. aim to determine the role of fl exible bronchoscopy with bronchial alveolar lavage (bal) in the management of patients with febrile neutropenia. methods a retrospective analysis was made of the number of patients admitted with febrile neutropenia at a single institution who underwent bronchoscopy plus bal from years to . computer database plus patient case notes were reviewed to establish clinical symptoms and signs, radiological fi ndings, antimicrobial treatment and mean duration to bronchoscopy following admission. results a total of episodes of febrile neutropenia were recorded years to . seven patients ( males and females) were referred for bronchoscopy plus bal. the mean age was . years (age range - years) and all had been diagnosed with acute lymphoblastic leukemia. all patients had at least cough as a clinical symptom along with radiological fi ndings. all patients had been on broad spectrum antibiotics at the time of bronchoscopy. the mean duration from admission to time of bronchoscopy was hours ( days) with a standard deviation of hours. of the seven patients one patient yielded a positive result on bal. this did not result in a change in management as the patient improved clinically before the result of the bal was confi rmed. conclusion in this retrospective case series the diagnostic yield of fl exible bronchoscopy plus bal in children with febrile neutropenia was low. prospective studies plus early timing towards bronchoscopy and bal should be conducted to further defi ne its role in the management of febrile neutropenic patients. confl ict of interest nil. methods prospective cohort study involving monthly follow-up with caregivers. two years post enrolment, children undergo clinical and lung function assessment (fot). presence of bronchiectasis is determined by physician review and radiological confi rmation (when indicated). the frequency of pbb episodes is recorded over the study period. of children recruited to the cohort study to date, % ( / ) were male. the median age at recruitment was months (iqr , ). % of children had recurrent pbb. of the children who have had -year clinical follow-up, were able to perform fot and % ( / ) showed abnormalities (reactance above normal range.) % ( / ) with pbb have had subsequent physician diagnosis of bronchiectasis or csld. conclusion the burden of cough in children with pbb years after diagnosis remains high. ongoing clinical follow-up of this cohort of children with pbb should provide further insight into the likelihood of progression from pbb to csld and bronchiectasis. support financial markets foundation for children (for project), allen & hanburys and qcmri (for dw), nhmrc (for ju and ac). introduction national streptococcus pneumoniae (sp) serotype surveillance reports only culture positive cases from sterile sites but the yield from culture is low. polymerase chain reaction (pcr) is more sensitive in detecting sp in culture negative samples. aim to determine whether enhanced molecular surveillance in childhood empyema provides additional sp serotype information compared to national surveillance methods. methods pleural fl uid from children with empyema underwent culture and pcr to identify sp-targeting autolysin (lyta) and multiplex pcr to identify sp serotypes. national surveillance data were obtained from the national notifiable diseases surveillance system (nndss) for the same time period and age groups. results empyema: children, male, median age . (range . - . ) were recruited from april for months. sp was cultured in / ( . %) in blood and / ( . %) in pleural fl uid. sp was identifi ed by pcr in / ( . %). serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). one subject had serotypes and in a serotype could not be established. nndss: sp culture positive cases were reported. serotypes: , n = ( . %); , n = ( . %); a, n = ( . %); f a, n = ( . %); v/ a, n = ( . %); f/ a, n = ( . %); non-typeable, n = ( . %). other serotypes were reported in sp positive cases. signifi cant differences between empyema and nsdss data were identifi ed for serotypes (p < . ) and (p < . ). conclusions the proportion of serotypes and were signifi cantly higher in empyema fl uid using pcr. this disease model provides additional serotype information to national surveillance data. this has important implications in monitoring replacement serotypes following the introduction of new vaccines. funded by glaxosmithkline, belgium. h giddings , l seccombe , p rogers , a corbett , e veitch recent theories on the pathophysiology of parkinson's disease (pd) emphasize early brainstem involvement. furthermore various respiratory function abnormalities have been reported without consistent pattern. we sought to study the effects of idiopathic pd on respiratory function and ventilatory response to hypercapnoea and hypoxia. methods patients with a diagnosis of pd but no known respiratory disease were recruited. subjects underwent lung function testing including respiratory muscle strength, ventilatory response to hypercapnoea (with central respiratory drive (p )) and a hypoxic simulation (fio % cough is the most common symptom presenting to doctors. paediatric cough is associated with signifi cant morbidity for both children and their parents. the symptom of cough is associated with airway hyper-reactivity and is a dominant symptom of airway infl ammation. inhaled corticosteroids (ics) can reduce airway infl ammation and hyper-reactivity. the objective of this review was to evaluate evidence for the effi cacy of ics in reducing the severity of cough in children with sub-acute cough (defi ned as cough duration of - weeks). methods search was conducted by the cochrane airways group using cochrane methodology. all randomized controlled trials (rcts) comparing ics with a control group for treatment of sub-acute cough in children were considered for inclusion. search results were analysed using pre-determined criteria for inclusion. results two studies were eligible for inclusion in the review, however there were limitations in that the participants of both these studies were infants, post acute bronchiolitis illness, and cough duration at start of study treatment was ill-defi ned. children were included in the meta-analysis. there was no signifi cant difference between groups in proportion of children 'not cured' (primary outcome measure), with a pooled or of . ( % ci . , . ) (using intention to treat analysis). conclusions there is currently no evidence to support the use of ics in sub-acute cough in children. however, this systematic review is limited by the small number of studies available for analysis and the quality and design of these studies. further well-designed rcts are required to support or refute the effi cacy of treatment with ics in children with sub-acute cough. once obstructive sleep apnoea (osa) is diagnosed, a cpap implementation sleep study is traditionally performed to determine the pressure required to control the upper airway. however, since modern cpap machines store sophisticated control data we reasoned it may equally be possible to commence cpap via a 'best guess' iterative approach without compromising osa control or compliance. aim to compare the outcomes at months of patients commencing cpap after best guess with those commencing cpap after a cpap implementation sleep study. methods we retrospectively reviewed the records of all patients referred by respiratory physicians to our cpap clinic between march and march , and the two methods of starting cpap were compared. data collected included age, sex, bmi, respiratory disturbance index (rdi), cpap pressure commenced, fi nal pressure at months, cpap usage data and cpap clinic contacts. results patients were identifi ed, aged ± years, %male, bmi . ± . , with severe osa, rdi ± . commenced cpap via best guess and after a cpap sleep study. the starting pressures in both groups were similar, . ± . versus . ± . cmh o. in those patients continuing to use cpap at months, there were no differences between the groups for fi nal pressure, numbers of patients changing pressure, control of osa with cpap, and hours cpap used per day. in the best guess group however, signifi cantly more patients were continuing to use cpap at months, % versus % (p = . ). conclusion this study demonstrates that it may no longer be necessary to perform cpap implementation sleep studies routinely and this will save hospital bed days. confl ict of interest nil. six required intubation and the rest were managed with non-invasive ventilation in icu. the average length of stay in icu was . days. polysomnographic data will be described. conclusions obesity hypoventilation as a cause of respiratory failure is likely to increase in frequency as the incidence of obesity increases. increased awareness by the lay public, as well as clinical suspicion and recognition of the condition by all clinicians at an earlier stage, is likely to prevent progression to the point of needing intensive care. it is hoped that this case series may provide a springboard for further study into why these patients presented at such a late stage of their disease process. supported by none. confl ict of interest none. although sa and sleepiness often co-exist, the commonest cause of sleepiness in a general community is depression, with sa being the th most common cause. in order to assist recognition of depression in a snoring population attending a sleep clinic, we introduced a simple two question 'beyond blue questionnaire(bbq)' into our routine assessment. aims to ( ) background indices of ventilation distribution in diffusion (s acin ) and convection (s cond ) dependent airways derived from multiple breath nitrogen washout (mbnw) may vary between interpreters because of differences in calculation of phase iii slopes (Δphase iii ). aims to compare s cond and s acin results of interpreters from a single mbnw in copd subjects. methods subjects with copd underwent mbnw. three washouts were analysed independently by experienced and novice interpreters using custom software for automated breath identifi cation. Δphase iii was fi tted automatically by least squares fi t between predetermined points, and then adjusted manually. s cond was the linear slope of Δphase iii plotted against lung turnover (cumulative expired volume/frc), between turnovers . - . s acin was the Δphase iii of the fi rst breath minus the s cond component. differences expressed as icc and cov, were examined by repeated measures anova. results mean ± sd age was ± years. fev was ± % predicted. s cond was greater while s acin was lower from the experienced introduction β-blockers may cause bronchoconstriction and mask the effect of β -adrenergic agonists. this has implications for the interpretation of routine diagnostic spirometry and bronchodilator response. this study examined this issue in a routine lung function laboratory, and whether it applied to both cardio-selective (c) and non-selective (nc) preparations. method all patients attending the lung function laboratory, royal adelaide hospital over a -month period were asked whether they were currently taking a β-blocker and to identify the drug. spirometry results were analysed to assess airfl ow obstruction and reversibility. results patients completed the survey and patients ( %) were taking β -blockers. the table shows the results of the patients who could be assessed for reversibility in spirometry. of the patients in this group patients ( %) were taking (c) and ( %) (nc) agents. fifty-three patients were unsure whether they were taking a β -blocker. no signifi cant differences were found in the percentage of patients with airfl ow obstruction or reversibility between the groups. aim to examine patterns of adult lung function in terms of airfl ow obstruction, hyperinfl ation and/or reduced diffusing capacity (d l co). this can then be related to the life-time history of risk factors such as smoking, asthma and infections. methods using the population-based tasmanian longitudinal health study (tahs) cohort followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (uncorrected for haemoglobin) and lung volumes was performed, then lung function data were analysed using the mean predicted values. airfl ow obstruction was defi ned as post-bronchodilator fev /fvc (post-b.d. fer) < . , hyperinfl ation as total lung capacity (tlc) > % predicted, and reduced d l co as < % predicted. aim to examine the gender-specifi c differences in adult spirometry, d l co and lung volumes, with a view to relating them to life-time respiratory risk factors. methods using the population-based tasmanian longitudinal health study (tahs) followed since , an asthma-enriched sub-sample was selected consisting of % ever with asthma, of whom half reported current asthma. measurement of spirometry, d l co (corrected for haemoglobin) and lung volumes were performed. data were analysed using the statistical upper and lower limits of normal of reference equations by nhanes iii, roca et al and quanjer et al. of the caucasian adults ( females), % completed all tests. mean age . years (range - ). elevated rates of airfl ow obstruction and hyperinfl ation were seen. signifi cantly higher proportions of females than males had reduced d l co and d l co/v a (p < . ). only . % (n = ) of females had a low d l co with low fev /fvc ratio, and . % (n = ) had a reduced tlc overall. there were no signifi cant gender differences in v a , tlc, or ever and current active smoking. males and females averaged over kg more than the mediterranean adults described by roca et al., however weight is not relevant to d l co in males. conclusion a higher percentage of middle aged females have a reduced d l co and/or d l co /v a, compared to males, with an increased rate overall. grant support nhmrc, australian postgraduate association. d chapman , , , j kermode , , , n brown , , , n berend , , , g king , , , background during bronchoconstriction, a deep inspiration (di) dilates the airways, which then re-narrow once tidal breathing is resumed. re-narrowing occurs faster in asthmatic subjects and may be due reduced airway distensibility. aim to determine the association between baseline airway distensibility and the rate of re-narrowing after di. methods eleven asthmatic and fi ve non-asthmatic subjects had baseline airway distensibility measured by forced oscillation technique (fot). after methacholine challenge, respiratory system resistance (rrs) was measured during min of tidal breathing, followed by di to total lung capacity (tlc) and passive return to normal tidal breathing. dilatation was measured as the decrease in rrs between end tidal inspiration and tlc, and re-narrowing as end-expiratory rrs immediately after di, as per cent rrs at end-tidal expiration before the di. distensibility is presented as geometric mean ± %ci and re-narrowing as mean ± % ci. results airway distensibility was reduced in asthmatic compared to healthy subjects ( . s − .cmh o − ( . - . ) vs. . s − .cmh o − ( . - . ), p = . ). dilatation did not differ between groups (p = . ) but re-narrowing was increased in asthmatic compared to healthy subjects ( ± % vs. ± %, p = . ). airway distensibility did not correlate with airway re-narrowing (r s = - . , p = . ). conclusion the increased re-narrowing after di in asthmatic subjects is not due to reduced baseline airway distensibility and may be due to increased shortening velocity of airway smooth muscle or reduced elastic recoil. supported by the nhmrc and the crc for asthma and airways. nomination nil. confl ict of interest no. c ng , , , s jenkins , , , n cecins , , p eastwood , , aim to evaluate the measurement properties of two accelerometers: the activpal and the stepwatch activity monitor (sam) in people with copd. methods the activpal and sam were attached to the anterior right midthigh and the right ankle, respectively (as per device recommendations). each participant performed walking tasks; at a self-selected slow speed and at a self-selected normal speed. at each speed, one walk was performed with a -wheeled walker (ww) and the other without. results participants aged ( ) years (fev = ( ) % pred; males) completed the study. the slow and normal speeds were ( ) m·min − and ( ) m·min − , respectively. agreement between steps recorded by the sam with steps counted during observation did not differ with speed or ww use (p = . ). the mean difference was steps·min − and the limit of agreement (loa) was steps·min − . agreement between steps recorded by the activpal with steps counted was worse at slow speeds (mean difference steps·min − with loa of steps·min − ) compared with normal speeds (mean difference steps·min − with loa of steps·min - ) (p = . ), but was not affected by ww use. both accelerometers detected the small difference in walk speed irrespective of ww use (p < . ). conclusions neither the accuracy nor responsiveness of either accelerometer was affected by ww use. in contrast to the activpal, sam was accurate at both speeds and therefore can be used to detect steps in people who walk very slowly during daily life. breathing and sleep, heidelberg vic., eastern health, melbourne vic., northern health, epping vic., and monash university, clayton vic. aim to document the care and pathways patients with copd travel at three metropolitan health services. methods data were extracted from data sets for patients attending the emergency department of the three hospitals with a diagnosis of copd over year. the three hospitals included a city-based tertiary/quaternary hospital and two smaller community hospitals. analysis was completed on similarities and differences in admission and referral rates, average length of stay, and discharge destination, standardized by age, sex and mode of transport to the emergency department. results there were inpatient separations and emergency department presentations for patients with copd. discharge patterns related to the designated role of the hospital, with the community hospitals discharging to % of patients directly home and the more specialized city hospital discharging % to other hospitals and % home. there were signifi cant differences in the admission rates for category and patients among the hospitals. we found unexplained variation in the acute average lengths of stay of . , . and . days. conclusions the analysis confi rmed some expected patterns based on the type of hospital, but also identifi ed unexplained variation that suggests that factors other than patient characteristics may be contributing to the variation in care pathways. aims to: ( ) determine which tests of exercise capacity relate to average daily energy expenditure (dee) and; ( ) quantify the intensity at which activities of daily living (adl) are undertaken in people with chronic obstructive pulmonary disease (copd). methods a study was undertaken in subjects with stable copd (mean, sd) aged ( ) years with an fev of ( ) % predicted ( males). measures were collected of distance walked during the six-minute walk test ( mwd) and incremental shuttle walk test (iswd) and peak rate of oxygen uptake during a cycle ergometry test (vo peak ). the sensewear armband® was worn during the waking hours for . ( . ) days to measure dee. the intensity at which activities of daily living were undertaken was expressed as a percentage of vo peak . results dee was associated with mwd (r = . ; p = . ), iswd (r = . ; p = . ) but not vo peak (r = . ; p = . ). stronger associations were observed between dee and the body weight-walking product for mwd (r = . ; p < . ) and iswd (r = . ; p < . ). the average intensity of adl was equal to ( %) of vo peak (range to %). conclusions mwd and iswd, but not vo peak were related to dee. as adl were performed at a high percentage of vo peak it may be more realistic to increase dee by increasing the frequency or duration, rather than the intensity of physical activity. in patients with copd, two mwts are recommended prior to commencing a pulmonary rehabilitation program (prp) to allow for a learning effect. aim to determine the characteristics of patients with copd in whom -minute walk distance ( mwd) did not increase on a second test. methods patients ( males) with stable copd (aged , to years) naïve to the mwt performed two tests ( minutes apart) prior to commencing a prp. patients were categorized according to their change in mwd with test repetition. results mwd was the same or decreased on the second test in patients ( %) (table) . in the remaining patients ( %), mwd increased by m ( %) ( % ci to m, to %). logistic regression analysis identifi ed fev (l) as the only signifi cant variable (p < . ) that predicted the absence of a learning effect in mwd with test repetition. conclusions some patients with severe copd may not require a practice mwt to achieve their maximum performance at a prp baseline assessment. ( ) years, with stable ipf were evaluated in this study. demographic data and measures of pulmonary function (spirometry, diffusing capacity for carbon monoxide, (dl co )), dyspnoea (baseline dyspnoea index, bdi), peripheral muscle force (isometric quadriceps force (qf) and handgrip force (hf)), functional exercise capacity ( -minute walk distance, mwd), limitation in daily activities (activities of daily living (adl) score), and health status (sf- ) were assessed. relationships between mwd and mrc grade, pulmonary function, qf, bdi and adl score were examined. results the number of subjects in mrc grades , , and was ( %), ( %), ( %) and ( %), respectively. pulmonary function, bdi, qf, hf, mwd, adl score, and sf- decreased signifi cantly with increasing mrc grade (all p < . ). moderate to strong correlations were found between mwd and mrc grade (r = − . ), dl co (r = . ), qf (r = . ), bdi (r = . ) and adl score (r = . ) (all p < . ). conclusions these fi ndings suggest that the mrc dyspnoea scale can be used to discriminate and classify subjects with ipf according to the severity of impairment and disability. ( ) year (mean, sd) completed two assessment sessions on separate days. on one day, they exercised twice to symptom limitation (tlim) on a treadmill. on the other day, they exercised twice to tlim on a cycle ergometer. the order of exercise modality was randomized between days. on both days, the only difference between the exercise tests was that bipap, titrated to patient comfort, was used during the second test. measures were made of; ) tlim and, ( ) the difference in dyspnoea, using borg scores, at tlim during the fi rst test and the equivalent exercise time during the second test (i.e. iso-time). results bipap increased tlim on the treadmill ( ( ) seconds; p = . ) but not the bike ( ( ) seconds; p = . ). the reduction in dyspnoea at iso-time on the treadmill and bike was similar being, ( ) and ( ), respectively (p = . ). conclusions bipap may confer greater benefi t in exercise tolerance exercising on a treadmill compared with a cycle ergometer in patients awaiting lung or heart-lung transplant. infection with rhinovirus (rv) is known to trigger acute exacerbations in subjects with asthma and these subjects also have increased susceptibility to the effects of rv. the mechanisms remain poorly understood, but appear to involve a host innate immune defect in the airway epithelium. aim we sought to determine in bronchial epithelial cells (becs) if oxidative stress in the form of exposure to cigarette smoke extract (cse), hydrogen peroxide (h o ) and eosinophil peroxidise (epo) results in impaired mitochondrial function and if this directly impairs signalling of rv infection through mda and alters the release of type i and type iii interferons (ifns). methods pbecs were grown to confl uence. cells were then exposed to cse ( %, no fi lter) or h o ( . mm) or epo. cells were then infected with rv -b (moi = ). virus replication was measured by cell titration assay. following infection, il- , cxcl- , cxcl- was measured using cytometric bead array and fl ow cytometry. supernatants and whole cell lysates were collected for ifn-β, bax and mda detection by western blot. ifn-λ and cytochrome-c was measured using conventional elisa. cell viability was assessed by annexin v-pe staining and fl ow cytometry. results rv infection alone induced cxcl- , il- , cxcl- and ifn-λ. pbecs treated with each of the oxidative stressors had increased cytochromec release and increased apoptosis. this mitochondrial dysfunction led to degradation of mda expression and resulted in specifi c suppression of cxcl- and ifn-λ. conclusions exposure of becs to an oxidative stress results in mitochondrial dysfunction in airway epithelial cells. this leads to defective antiviral signalling in the airway epithelium after infection with rv. introduction pleural infection is associated with high morbidity. prompt drainage is key, but pus is often loculated and thick making drainage diffi cult. based on promising animal studies, we hypothesize that intrapleural therapy with t-pa and dnase, which lyse adhesions and reduce fl uid viscosity respectively, can signifi cantly improve pus evacuation in pleural infection. methods consecutive patients with pleural infection were treated with standard antibiotics and intercostal chest tube (ict) drainage. additionally, t-pa mg and dnase mg (each in ml of . % nacl) were instilled intrapleurally via an ict twice daily for up to six doses. the ict was clamped for minutes after each instillation. patients were followed clinically and with serial cxr. opacity from pleural effusion was quantifi ed on chest radiographs. results eleven patients ( male; mean age ) were treated. nine effusions were associated with community acquired pneumonia, of these, eight were visibly purulent, fi ve were culture positive and the mean fl uid ph was . (range . - . ). ten patients ( %) were successfully managed conservatively and one patient required surgery. median hospital stay from fi rst intrapleural treatment dose to discharge was days (range - ). the median amount of fl uid drained in the hours preceding t-pa/dnase treatment was ml (range - ), and improved signifi cantly to ml (range - ) following two doses of treatment. this was paralleled by a signifi cant reduction in radiographic opacity by a mean value of % of the hemithorax (range - %). four patients showed an initial rise in crp following t-pa/dnase, but all patients had resolution of sepsis and signifi cant reduction in crp. there were no major complications. pleuritic chest pain requiring opioid analgesia developed in three patients. methods clinical data were collected using a standardized form for aboriginal children aged days -< months hospitalized with alri and enrolled in a rct of vitamin a/zinc supplementation were matched with data collected during a population-based study of who-defi ned primary endpoint pneumonia (who-p). sensitivities, specifi cities, positive and negative predictive values (ppv, npv) for these signs were compared between who-p cases and lobar pneumonia assigned by a respiratory paediatrician. in episodes of hospitalized alri, who-p was diagnosed in ( . %); the respiratory paediatrician classifi ed ( . %) as lobar pneumonia. the sensitivities of clinical signs ranged from a high of % for tachypnoea to % for fever + tachypnoea + chest-indrawing; the ppv range was % to %, respectively. higher ppvs were observed against the paediatric respiratory physicians diagnosis compared to who-p. conclusions clinical signs on admission are not useful in predicting who-p in this population, presenting challenges for future pneumonia research in this population. who-p may underestimate alveolar consolidation in a clinical context and its use in clinical practice or in research designed to inform clinical management in this population should be avoided. the incidence of tb in the non-indigenous australian population is uncommon at . cases per population . in this paper, we report three cases of pulmonary tuberculosis in young australian born, non-indigenous adults in the hunter new england area where marijuana possibly was a signifi cant risk factor in transmission and severity of disease. all three cases had severe cavitating disease at time of presentation. contact tracing from the fi rst case, a regular heavy marijuana user, identifi ed mantoux positive contacts, one of whom developed active pulmonary tuberculosis. all contacts, mainly young adult males, denied sharing marijuana with the index case. contact tracing from the second case identifi ed mantoux positive contacts, of whom use marijuana regularly and shared bongs (water pipes) with the index case. there were positive mantoux contacts of the third case, one of whom shared bongs with the index case. health professionals need to remain aware of the possibility of tuberculosis in groups with historically low incidence rates. marijuana bong smoking is possibly associated with transmission and severity of tuberculosis . introduction in , these previously well women survived and made a good recovery from severe pneumonia and acute lung injury after retrieval on ecmo. streptococcus pyogenes is an unusual cause of pneumonia in adults. case a -year-old veterinarian with a history of mild asthma presented with days of fever and respiratory symptoms. the diagnosis was confi rmed by a fourfold rise in the anti-streptococcal antibody. this was complicated by respiratory failure, septic shock, acute renal failure, severe pulmonary hypertension and bilateral parapneumonic effusions. despite maximal interventions she deteriorated. femoral venous-venous ecmo was initiated on day at the calvary mater hospital in newcastle by a retrieval team from royal prince alfred hospital (rpa), sydney. she was transferred kms on ecmo in a large multipurpose ambulance. she developed lung abscesses and recurrent pneumothoraces and she required a pleurodesis. she required days of ventilation and days of ecmo. three months later she was asymptomatic, with mildly restrictive spirometry and minor cxr change. case a -year-old offi ce worker with s pyogenes bacteraemia made a similar presentation to our institution. she was ventilated for days, ecmo was initiated by the retrieval team and continued for days. three months later she was asymptomatic with a normal cxr and pulmonary function tests. introduction the urinary pneumococcal antigen (upa) test has been shown to have superior sensitivity to other investigations in determining the aetiology of community-acquired pneumonia (cap), but there is very limited data on its performance in local populations. the aims of this study are to establish the prevalence of positive upa testing in patients admitted to hospital with cap, and determine its utility. secondary aims are to identify associations with positive testing, as well as to determine if a positive test infl uences clinical outcomes. methods the study is a prospective, single-centre study that is still recruiting. adult patients are included upon admission to hospital if they have the diagnosis of cap, as defi ned by new infi ltrates on chest radiograph along with consistent clinical features. clinical data including curb- score of severity, current and prior antibiotics, co-morbidities, mortality and length of hospital stay are recorded. results preliminary results show a positive test prevalence of / ( . %, % ci . - . %) amongst patients admitted with cap. overall prevalence of pneumococcal pneumonia is / ( . %, ci . - . %). patients with a positive upa result have a higher mean curb- score of . compared with . in those with a negative result (p = . ). . % of patients with a positive result were admitted to the intensive care unit, compared with . % those with a negative result (p = . ). conclusions the overall prevalence of positive upa testing in patients admitted to hospital with cap is low. preliminary data suggests that patients with positive results are more likely to have greater severity pneumonia and to require intensive care support. comparative data on length of stay, mortality, previous antibiotic use and specifi c co-morbidities has not revealed any statistically signifi cant differences between positive and negative groups. confl ict of interests no. s herath , c lewis , m nisbet , respiratory department, auckland city hospital, auckland, new zealand, and infectious diseases department, auckland city hospital, auckland, new zealand rhodococcus equi (r. equi), previously known as corynebacterium equi is a gram positive bacillus that is found in soil and causes infection in grazing livestock. it is infrequently isolated from clinical specimens. it is usually associated with human disease in immunocompromised patients and is an uncommon cause of infection in immunocompetent patients. infection is usually acquired by the airborne route with pneumonia being the most common manifestation but it can also be acquired orally or by direct inoculation. we present a case of pneumonia caused by r. equi infection in a year old male builder who presented with cough, dyspnoea and night sweats. r. equi was cultured from a transbronchial aspirate from a subcarinal lymph node. despite extensive investigation, no contributing host immune defect was identifi ed. the patient recovered after three months of antibiotic treatment, initially with intravenous vancomycin and meropenem followed by oral clarithromycin and rifampicin. although infections due to r. equi have been increasingly reported in immunocompromised patients, since there have only been cases described in patients where no associated host immune defect was reported. in this cohort, the median age at presentation was years (range - ) and ( %) patients were male. ten ( %) of these cases had pulmonary infection. two ( %) patients died and the remainder were successfully treated with prolonged antibiotics. r. equi is an uncommon cause of infection in humans and rarely occurs in patients where a host immune defect cannot be identifi ed. introduction recognition of pulmonary involvement in extra pulmonary tuberculosis (ep-tb) may be an important public health issue, as it has been estimated that patients with smear negative pulmonary tb (ptb) are responsible for % of new infections. usually, all patients with ep-tb have a chest x-ray but sputum cultures are requested only if there is an abnormality. methods in this retrospective clinical audit, we aimed to evaluate the percentage of ep-tb patients with ptb despite a normal chest x ray (cxr), and to explore any clinical characteristics of this group. clinical notes, microbiology and cxr reports were reviewed from consecutive patients presenting with ep-tb between and . results of patients with ep-tb, % were male and the mean age was (range to ). most patients were of asian ethnicity (n = , %). the commonest presentation of ep-tb was lymphadenopathy (n = , %), followed by pleural (n = %) and bone (n = , . %) disease. ep-tb was diagnosed by biopsy/excision of the ep site in the majority (n = , . %), and by sputum testing alone in ( . %). sputum cultures were performed in n = , ( %) overall, with n = ( %) being positive. there was higher infl ammatory markers in the sputum culture positive group (esr . vs. . , p = . and crp . vs. . , p = . ). the majority had cxr abnormalities (n = , %). in the group with normal cxr (n = ), ( %) had sputum cultures performed. of these, were culture positive and of these also + smear positive ( on immunosuppression, with cough). conclusion a small number of patients with ep-tb and normal cxr had pulmonary tb, of whom were smear positive. thus, induced sputum testing should be considered in patients with ep-tb even if cxr is normal. this may aid diagnosis and determine infectivity. ntm are normal inhabitants of environmental reservoirs including water. disease due to ntm has been increasing in qld. aim to document the presence of ntm in potable water in brisbane, to compare the species isolated during summer and winter and to relate this to the geographic distribution of patients with ntm. methods water samples ( l) were collected from routine collection sites in winter and sites in summer . samples were processed in triplicate as previously described. h subcultures were taken from positive specimens, dna extracted, followed by s rrna sequencing. patient addresses were obtained from the qld tb control centre database. aim to gauge the full impact of pandemic h n infl uenza across demographic groups in the northern territory, particularly indigenous and remoteliving individuals. methods we performed two cross-sectional serological surveys on specimens from residents of the northern territory, with specimens obtained from january to may (pre-pandemic) and specimens from september (post-pandemic). specimens were selected from among serum tubes collected from ambulatory outpatients. antibody titres were measured by haemagglutination inhibition against the a/california/ / reference virus. all specimens had available data for gender, age, and address, with indigenous status determined in . % of cases. results protective antibody levels, defi ned as a titre of or greater, were present in . % of pre-pandemic specimens and . % of post-pandemic specimens. the pre-pandemic proportion immune was greater with increasing age, but did not differ by other demographic characteristics. the post-pandemic proportion immune was greater among aboriginal and torres strait islanders and in younger age groups, but did not differ by gender or socio-economic index for area. however, the proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups. the northern territory-wide attack rate adjusted to age, region and indigenous status was . %. conclusions pandemic infl uenza disproportionately affected children and indigenous australians in the northern territory in . the proportion of specimens demonstrating post-pandemic immunity was particularly variable among indigenous and remote-living individuals. the kormp found asymptomatic aboriginal children (ac) had more hrv than asymptomatic non-aboriginal children (non-ac) in a longitudinal communitybased cohort study where infants had nasopharyngeal aspirates (npa) collected regularly from birth to years of age. aim to compare the frequency of hrv groups in asymptomatic ac and non-ac in the kormp. methods npa positive for hrv (n = ) from the npa previously tested for respiratory viruses, had viral rna extracted and reverse transcribed. hrv was detected and typed using a two-step pcr of the hrv ' utr, followed by dna sequencing for typing. chi-square analyses were used. results hrv was detected and typed in npa (from children; ac and non-ac), could not be typed and were not positive for hrv. ac had more hrv in summer and autumn than non-ac and were more likely to be co-infected with at least / bacterial species identifi ed. hrva, b & c were found in . , . and . % of hrv detected. hrvb & c were increased in infants exposed than not exposed to tobacco smoke in utero (hrvb; . vs. . %, p = . and hrvc; . vs. . %, p = . ). of the npa, hrv-a was detected more often in npa from ac than non-ac ( . vs. . %, p = . ), particularly at - months of age (p = . ) and during summer (p < . ). hrvb was detected more often in npa from ac than non-ac in autumn (p < . ). hrvc was detected as often in ac as non-ac in each season except summer. aim to determine whether interferon-gamma release assay (igra) can be effectively used for diagnosis of latent tuberculosis infection in a remote location. methods subjects were enrolled from the darwin centre for disease control tuberculosis clinic and were eligible if a tuberculin skin test (tst) of mm or greater had been recorded for any indication. igras were performed using quantiferon®-tb gold whole blood in-tube assay according to manufacturer's instructions. specimens were incubated and centrifuged at the local laboratory before refrigeration for transport. interferon assay was performed at the reference laboratory, over km away. results igras were performed, with patients ( %) recording negative results, ( %) positive and only one result ( %) indeterminate. negative, and therefore discordant, test results were more common in bcg vaccinated individuals. this effect was not limited to those with tst results of - mm, but was seen primarily in those with results of mm and above. conclusions these results are broadly comparable to fi ndings for igra use in less remote settings. in particular, our low rate of indeterminate results suggests that igra testing is feasible at a remote site after local processing. this approach could be considered for use in the northern territory tuberculosis control program. inhaled medications form the mainstay of drug treatment for patients with airways disease. effectiveness of therapy is dependent on the appropriate selection and prescription of drug and device, correct supply and adherence to therapy with an effective technique. patients frequently admit to acute medical wards both with acute exacerbations and for other co-morbidities eg heart failure or pneumonia. inpatient episodes provide an opportunity to review inhaled therapy however anecdotally add to patient confusion and introduce complexity (rational or ad hoc changes to inhaled drug, device, strength, dose or frequency). aim identify prescribing accuracy and effectiveness of patients' inhaler technique. describe any discrepancies between inhaled therapy: ( ) used prior to admission, ( ) prescribed for inpatient use, ( ) available at the bedside and ( ) administered, prior to and after implementation of an inhaler prescribing and administration guide. methods a single day audit of all inpatients on general medical wards was conducted october (review of medication charts and inhalers in patients' bedside lockers, brief questioning and direct observation of patients' inhaler technique. results compared to post implement of the 'prescribing and administering inhalers' tool (audit in december ). results from ( %) patients had inhalers prescribed, (mean: . prescriptions per patient). % of prescriptions were accurate ( % patient had no errors). discrepancies between used prior to admission and inpatient prescriptions were found in ( %) patients while those between inpatient prescriptions and available at the bedside were found in %. self-administration ('s') was noted on medication charts of ( %) patients, of whom had an ineffective inhaler technique. / patients has a spacer at the bedside with a further r prescribed metered aerosol inhalers. post-intervention differences in prescribing, supply, administration and technique errors will be discussed. conclusions a combination of errors and prescription discrepancies reduce the effectiveness of inhaled therapy for inpatients. confl ict of interest no. males (n (%) % ci) females (n (%) % ci) adm and bed days bmi, body mass index hrqol, health related quality of life chronic respiratory disease questionnaire; adm, admissions, mean (sd) uberculosis notifi cations in australia a cluster of tuberculosis associated with use of a marijuana water pipe the prince charles hospital foundation cc dobler , , gb marks , woolcock institute of medical research, the university of sydney, nsw, and department of respiratory medicine, liverpool hospital, sydney, nsw aim to determine the incidence rate and nature of adverse events in patients taking treatment for latent tuberculosis infection (ltbi). methods records of all patients who received treatment for ltbi at the chest clinic of a large tertiary hospital between / and / were reviewed. an adverse event was defi ned as any change in health status or side effect that led to treatment interruption or cessation. liver function tests were not performed routinely during follow-up, except when the patient was considered to be at an increased risk of developing hepatitis. results of patients in whom treatment for ltbi was initiated ( %) received isoniazid for months, ( %) received a combination of isoniazid and rifampicin for months, and the remainder were treated with different regimens. their mean (sd) age was ( ) years and % were male. nineteen patients ( . %) experienced an adverse event. seven patients developed a rash, four had lethargy and/or mood disorders, three had subclinical hepatitis, four experienced severe nausea, vomiting and/or other gastrointestinal symptoms and three had features of peripheral neuropathy. in eight patients who experienced an adverse event medication was temporarily ceased and then re-started without change; in four the treatment regimen was changed; and in seven the treatment was ceased completely. the risk of adverse events was not signifi cantly related to age, sex, drug regimen (single drug versus combination therapy) or baseline transaminase levels. conclusions in this cohort almost in patients on treatment for ltbi experienced an adverse event. although the adverse events were generally mild to moderate, this risk has to be taken into account when deciding whether to advise treatment for ltbi. introduction human rhinovirus (hrv) is the commonest cause of asthma exacerbations in children. pernasal aspirate (pna) is the gold standard for microbiological sampling but is invasive and distressing for children. studies have showed that less invasive swabs may be just as effi cacious. aim to test the hypothesis that hrv detection is as effi cient using nasal fl ocked swabs or washes and more comfortable, compared with pna in children with respiratory illnesses. methods children were recruited on presentation to the emergency department with respiratory symptoms. pna was collected from one nostril of all children recruited and nasal fl ocked swab (n = ) or wash (n = ) collected from the other nostril alternately. subjects rated the comfort of each sampling method to (least to most). viral rna was extracted and reverse transcribed. a two-step pcr of the hrv ' utr was used for detection, followed by sequencing for typing. results to date, children ( % male, mean age of . years) had paired samples taken. of these children, % (n = ) presented with a diagnosis of viral induced wheeze and % (n = ) had a hrv positive sample. compared with pnas, nasal fl ocked swabs were % ( of pna positive) effective in detecting hrv, whilst nasal washes showed % ( of pna positive) effi cacy. of the successfully typed samples, had hrva and had hrvc. nasal washes had a better comfort rating (mean . , n = ) than fl ocked swabs (mean . , n = ) and pnas (mean . , n = ). conclusion our fi ndings suggest that whilst nasal fl ocked swabs are an effective sampling method for hrv detection, nasal washes were more effective, being as effective as pnas and were the most comfortable. support nhmrc, pmh foundation. nomination nil. aim to describe the inpatients treated by a dedicated niv service. methods a retrospective audit of inpatients treated by the alfred niv service between january and june . the defi nition of niv included patients treated with cpap and bilevel positive pressure ventilation. results patients (age: ± years (mean ± sd), gender: % male) were treated with niv on occasions (repeat admissions patients). commonest indications for niv were osa (n = , %), acute exacerbations (ae) of copd (n = , %), acute cardiogenic pulmonary oedema (acpo) (n = , %) and post-lung transplantation (n = , %). treatment was delivered primarily in the respiratory ward (n = , %), cardiac ward (n = , %), icu (n = , %) and general medical ward (n = , %). episodes of cpap (mean pressure ± cmh o), osa and acpo made up % of those treated. seventy-two episodes of bilevel pap (mean ipap ± cmh o and epap ± cmh o), aecopd and weaning post-mechanical ventilation made up % of those treated. outcome data was available in a subgroup of patients with acpo (n = ) andaecopd (n = ). in the acpo group, patients ( %) improved and niv was ceased. three patients ( %) deteriorated and were intubated and patients ( %) were palliated. in the aecopd group, patients ( %) improved andniv was ceased or they were discharged on therapy. patients either deteriorated on niv or could not tolerate therapy, of these ( %) continued ward management and ( %) were palliated. conclusion the alfred niv service model has managed a large number of referrals across a range of diseases in a variety of wards. this is likely to have reduced demand on icu, hdu and respiratory ward beds. compared to the published literature, theoutcomes for acpo are worse than expected but comparable for aecopd. this may be explained by local referral patterns for acpo. we believe that our service model provides a viable means of administering niv to an ever expanding referral base. transitional & community service, the university of south australia, adelaide, sa , the university of adelaide, adelaide, sa, , the mary potter hospice, north adelaide, sa, , thoracic medicine, the royal adelaide hospital, adelaide, sa, , the royal district nursing service, wayville, sa , and the palliative care council of sa eastwood, sa introduction: the adelaide health service is in the process of developing a new and innovative model of copd community based care. a number of initiatives have informed this development including a recent research project examining the experiences of participants with end stage copd and their carers. a growing body of evidence indicates the importance of a palliative approach, however this often takes the form of referral to a palliative care service rather than a broader application of palliative principles in both specialist and primary care. methods: fifteen participants were interviewed twice at monthly intervals to explore their needs and the services they accessed. a series of focus groups with key service providers in sa was also undertaken. data were analysed to identify how hospital, specialist palliative care units and primary care services currently interface to meet identifi ed patient and carer needs. results: the current service model is episodic and reactive with services activated through the acute care system. our research has shown that, as copd advances, current models of care do not address the importance of supporting quality of life (including a focus on adls) and carers in their ongoing role. also emphasised was the lack of co-ordination of care, collaboration between service providers and communication -the basics of chronic disease management. conclusions the outcomes of this study will inform the development of a proactive, multidisciplinary model of care which is no longer reliant on tertiary care, but places primary care at the centre of the model. greater collaboration between respiratory, palliative and primary care services will provide an integrated approach, focusing on the needs of the patient and carer. aim long term conditions are prevalent in south auckland and impact on the individual, the community and the health system. as nurses living within this community, and employed by counties manakau district health board, our aim was to explore funding opportunities available through the pacifi c health team. lotumoui was established to improve health outcomes/behaviours for pacifi c populations. the church we attend has wide cultural diversity and had no knowledge of the programme and the support provided to make healthy changes within our community. methods firstly a health committee was formed within the church, having 'sold' our vision to the parish council. we launched the group by undertaking free blood pressure checks, followed by a 'walk the talk' project for the days leading into easter. baseline observations were taken and pedometers issued. results the parishioners who attend regular exercise sessions are reporting improved quality of life, exercise tolerance and reducing waist lines. bp parameters are also reducing. conclusions a dedicated health committee within a parish community, supported by the district health board can impact on changes in lifestyle by simple interventions. the investment by the pacifi c team will reap benefi ts for the individual and the health sector. confl ict of interest no. key: cord- -hth nf b authors: tsukagoshi, hiroyuki; ishioka, taisei; noda, masahiro; kozawa, kunihisa; kimura, hirokazu title: molecular epidemiology of respiratory viruses in virus-induced asthma date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: hth nf b acute respiratory illness (ari) due to various viruses is not only the most common cause of upper respiratory infection in humans but is also a major cause of morbidity and mortality, leading to diseases such as bronchiolitis and pneumonia. previous studies have shown that respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (hpiv), and human enterovirus infections may be associated with virus-induced asthma. for example, it has been suggested that hrv infection is detected in the acute exacerbation of asthma and infection is prolonged. thus it is believed that the main etiological cause of asthma is ari viruses. furthermore, the number of asthma patients in most industrial countries has greatly increased, resulting in a morbidity rate of around - % of the population. however, the relationships between viral infections, host immune response, and host factors in the pathophysiology of asthma remain unclear. to gain a better understanding of the epidemiology of virus-induced asthma, it is important to assess both the characteristics of the viruses and the host defense mechanisms. molecular epidemiology enables us to understand the pathogenesis of microorganisms by identifying specific pathways, molecules, and genes that influence the risk of developing a disease. however, the epidemiology of various respiratory viruses associated with virus-induced asthma is not fully understood. therefore, in this article, we review molecular epidemiological studies of rsv, hrv, hpiv, and hmpv infection associated with virus-induced asthma. acute respiratory illness (ari) is a major cause of morbidity and mortality worldwide (williams et al., ; sloots et al., ) . ari imposes a large burden on health, particularly in children. for community-based care, ari has been estimated at a cost of over us$ per case (ehlken et al., ) . the disease burden for ari is estimated at , , disability-adjusted life years and . million deaths (world health organization, ) . thus, ari has a huge impact on health and society. although severe lower respiratory tract infections have been observed, ari is most often associated with mild upper respiratory infection (uri). most ari cases in early childhood are confirmed as uri, leading to symptoms of the common cold with coryza and cough. in contrast, around one-third of infants with ari develop lower respiratory tract symptoms such as tachypnea, wheezing, severe cough, breathlessness, and respiratory distress (tregoning and schwarze, ) . in general, viruses are the most common causative agents of ari. more than different types of viruses are known to cause ari, with respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), and human parainfluenza virus (hpiv) most commonly identified in ari patients. indeed, together with these respiratory viruses, human enterovirus (hev), influenza virus (infv), human coronavirus (hcov), adenovirus (adv), and human bocavirus (hbov) account for around % of aris detected (kusel et al., ) . respiratory viral infections can have severe adverse outcomes in patients with established asthma and are associated with nearly % of asthma exacerbation episodes (nicholson et al., ; johnston et al., ; wark et al., ; heymann et al., ; grissell et al., ) . accumulating evidence indicates that the etiology of most cases of asthma, namely virus-induced asthma, is linked to such respiratory virus infections. in addition, rsv and hrv are the most frequently detected pathogens and may play an important role in viral induction and exacerbation of asthma. molecular biology techniques have developed rapidly over recent years. the application of molecular techniques to the study of virus-induced asthma enhances epidemiologic studies by improving our ability to classify these pathogens into meaningful groups (foxman and riley, ) . in this review, we focus on molecular epidemiological studies of respiratory viruses, including rsv, hrv, hmpv, and hpiv infections, associated with virus-induced asthma. in infancy, illnesses such as bronchiolitis share many clinical features with acute asthma, including wheezing, rapid breathing, prolonged expiratory phase inflammation, and respiratory www.frontiersin.org compromise. respiratory viruses are detected in the majority of asthma exacerbations in both children ( - %) and adults ( - %; johnston et al., ; grissell et al., ) . in addition, wheezing illnesses are also closely associated with respiratory viral infections in all age groups (gern, ) . fujitsuka et al. ( ) attempted to detect various respiratory viruses in japanese children with acute wheezing using pcr technology and found viruses in samples from . % patients: rsv or hrv alone were detected in . and . % patients, respectively and both rsv and hrv were detected in . % patients. other previous reports suggested that the prevalence of rsv and hrv is similar ( and %, respectively) in children less than years of age, but differs ( and %) in older children (johnston et al., ; grissell et al., ) . in addition, fujitsuka et al. ( ) suggested that rsv was the dominant species detected in patients with no history of wheezing and/or asthma, while hrv was dominant in patients with such a history. thus, the main causative viral agent of asthma depends on previous illness and age. around one-third of infants who have an acute wheezing illness go on to develop recurrent wheezing, indicating that viral respiratory illnesses in early life promote asthma. recently, the "two-hit" hypothesis has been proposed, whereby viral infections promote asthma mainly in predisposed children (gern, ) . infants who develop virus-induced wheezing episodes are at increased risk for subsequent asthma, but most acute wheezing illnesses in infancy resolve with no long-term sequelae. it has been recognized for years that rsv infections often produce the first episode of wheezing in children who go on to develop chronic asthma (lemanske, ) . indicators of heightened risk for developing asthma include wheezing episodes caused by hrv infections and the development of atopic features such as atopic dermatitis, allergen-specific ige for foods or aeroallergens (e.g., house dust, mites, or cat or dog dander), and blood eosinophilia (figure ) . once asthma has been established, hrv infections are the most common cause of figure | relationship between respiratory viral infections and development of asthma. host-pathogen interactions that determine the severity of respiratory illnesses, and risk for subsequent asthma was increased by respiratory virus infection, especially due to rsv, in infants. although most acute wheezing resolves within a relatively short time, a history of wheezing and host immunological conditions (e.g., atopic features) heightens the risk for asthma. once asthma is established, hrv infections are the most common causative agents of asthma in children. acute exacerbations, especially in children. as in infancy, atopy is an important risk factor for acute episodes of virus-induced wheezing (kusel et al., ) . many previous reports have suggested that such respiratory virus infections are deeply associated with virus-induced asthma (kusel et al., ; pierangeli et al., ; kuehni et al., ; fujitsuka et al., ; kato et al., ) . thus, it is entirely plausible that viral infections induction and/or exacerbation asthma in children. respiratory syncytial virus of genus pneumovirus and family paramyxoviridae causes ari in children (vardas et al., ; peter and james, ) . rsv infection may cause major problems in infants less than year of age and can lead to life-threatening aris such as bronchiolitis and bronchopneumonia (shay et al., ; leung et al., ; yorita et al., ) . epidemiological studies suggest that around % of infants have experienced an rsv infection by the age of year, and % by the age of years; host response to the virus varies greatly, but includes upper respiratory tract infections, typical bronchiolitis, and rsv-induced wheezy bronchitis (cane, ; kuehni et al., ). long-term prospective case-control and cohort studies have also linked rsv bronchiolitis to the development of wheezing and asthma later in childhood (sigurs et al., (sigurs et al., , (sigurs et al., , henderson et al., ) . thus, rsv infections may be associated with the initiation and/or exacerbation of asthma. the rsv genome encodes proteins (peter and james, ) . among these, the attachment glycoprotein (g) is a major structural protein that may be associated with both infectivity and antigenicity (johnson et al., ; rueda et al., ) . molecular epidemiological studies have shown that rsv can be classified into two phylogenetic subgroups, rsv-a and rsv-b (mufson et al., ) . the strains of subgroup a can be subclassified into eight genotypes (ga -ga and saa ), as can those of subgroup b (ba, gb -gb , and sab - ; parveen et al., ) . from phylogenetic analysis of the g gene of rsv, martinello et al. ( ) showed that rsv belonging to ga genotype may be associated with greater severity of illness in, for example, bronchiolitis and pneumonia. although ga genotype has been detected in the united kingdom, spain, and new zealand, it is not the most prevalent strain (cane et al., ; garcia et al., ; matheson et al., ) . martinello et al. ( ) therefore suggested that the association between greater severity of illness and ga genotype may be solely due to a transient shift in genotype-specific immune status within the community. in addition, correlations between certain strains and/or genotypes of rsv and slight differences in disease severity have been described previously (hall et al., ; walsh et al., ) . some genotypes such as subgroup a genotypes ga , ga , ga , ga , and na and subgroup b genotype ba have been detected throughout the world in recent years (zlateva et al., ; parveen et al., ; zhang et al., ; nakamura et al., ; rebuffo-scheer et al., ) . of these, na is a novel genotype known to be genetically close to ga genotype, while ga genotype and ba genotype are the most common genotypes of rsv subgroups a and b around the world and have persisted for many years (tran et al., ) . furthermore, a new genotype belonging to rsv-a, on , has been detected in some countries, including canada, korea, malaysia, south africa, and japan (eshaghi et al., ; lee et al., ; khor et al., ; tsukagoshi et al., ; valley-omar et al., ) . this genotype contains a unique tandem repeat ( nt sequence duplication) in the c-terminal rd hypervariable region of the g gene, and may be classified as a subdivision of na (eshaghi et al., ) . some reports have suggested that the severity of illness is not linked to subgroups or genotypes, but is associated with the quantity of rsv in nasopharyngeal aspirate (sullender, ; campanini et al., ) . a larger population study is needed to identify the different rsv genotypes circulating in different areas to gain a better understanding of the relationship between disease severity and rsv genotype. the g protein is a major antigen of rsv and amino acid substitutions may be related to changes in antigenicity. there are some reports of amino acid substitutions, and some positively selected sites in the c-terminal rd hypervariable region of the g gene have been estimated (botosso et al., ; yoshida et al., ; kushibuchi et al., ) . for example, yoshida et al. ( ) estimated some sites under positive selection in the region (asn ser, met glu, arg lys,and arg glu substitutions in rsv-a strains were estimated by the rel method, and asn tyr and leu pro substitutions of rsv-a, as well as leu pro substitution of rsv-b, were estimated by the ifel method). botosso et al. ( ) found and amino acid sites under putative positive selection in rsv-a and rsv-b, respectively. in addition, some unique positively selected sites were found in the g gene . these amino acid variations at these sites might play a key role in severe respiratory infection, such as bronchiolitis (goto-sugai et al., ) . furthermore, the rate of molecular evolution of the region might be high. for example, kushibuchi et al. ( ) estimated the evolutionary rate of rsv-a at . × − substitutions/site/year, while that of rsv-b was estimated at . × − substitutions/site/year. thus, it is suggested that this c-terminal rd hypervariable region in the g gene of rsv-a and -b evolved rapidly . based on host immunological conditions, it is suggested that host immunity such as tlr polymorphism is linked to symptomatic rsv infection (delgado et al., ) . thus, both the antigenicity of the viruses and host immune conditions may play important roles in the pathophysiology of severe respiratory infections such as bronchiolitis, pneumonia, and virus-induced asthma (awomoyi et al., ) . human rhinovirus are a group of positive-sense ssrna viruses belonging to genus enterovirus in the family picornaviridae (turner and couch, ) . although hrvs were previously thought to be mainly associated with the common cold causing mild respiratory symptoms, recent reports strongly suggest that hrvs may induce and/or exacerbate asthma (virus-induced asthma; chung et al., ; turner and couch, ; busse et al., ; gern, ; khadadah et al., ) . one report suggested that hrv wheezing illness within the first three years of life is significantly associated with the development of asthma at age years (jackson et al., ) . another report suggested that hrvs are major agents in the induction of wheezing and exacerbation of asthma (khadadah et al., ) . thus, hrvs are being re-evaluated as important agents of ari in humans (imakita et al., ; wos et al., ) . the basis for these lower respiratory symptoms has been a source of controversy in terms of the mechanisms of hrv pathogenesis. there are a variety of potential barriers to hrv infection of the lungs, including temperature-sensitive replication of the virus. for this reason, it is thought that the optimum propagation temperature of hrvs may be - • c in vitro (papadopoulos et al., ; schroth et al., ) . however, a recent study suggested that hrvs can propagate in lower airway tissues and this may be an important factor in the development of airway obstruction, coughing, and wheezing that can lead to bronchiolitis and pneumonia (mosser et al., ) . hrv has been concomitantly isolated with bacterial pathogens in - % of children and - % of adults with pneumonia (juven et al., ; templeton et al., ; jennings et al., ) . thus, it is not clear whether hrv is ever the causative agent for the disease. human rhinovirus were previously classified into two species, hrv species a (hrv-a) and species b (hrv-b), containing over serotypes (turner and couch, ) . however, a genetically heterogeneous third species, hrv species c (hrv-c), was discovered recently (lamson et al., ; mcerlean et al., ) . recent reports suggest that hrv-a, b, and c have a unique and wide genetic diversity simmonds et al., ; arakawa et al., ) . hrv-a and -c appear to be mainly associated with aris and virus-induced asthma, while hrv-b has been detected in a relatively small number of patients with aris (linsuwanon et al., ; wisdom et al., ; smuts et al., ) . our previous findings obtained from samples from children with aris in japan indicated that hrv-a and -c can be classified into many clusters in the phylogenetic tree, with % nucleotide divergence of the vp /vp coding region (mizuta et al., a; arakawa et al., ; kiyota et al., ) . in addition, kiyota et al. ( ) estimated that the rate of molecular evolution of the vp /vp coding region was rapid ( . × − substitutions/site/year) in hrv-c. these results suggest that hrv-a and -c detected in ari cases are the predominant strains and have varied genetic properties (wisdom et al., ; mizuta et al., a; arakawa et al., ) . thus, the association between hrv type and disease severity is not fully understood. there may be important differences in the susceptibility of individuals to the replication of hrv in lower airway tissues. parry et al. ( ) and gern et al. ( ) found that weak peripheral blood mononuclear cell (pbmc) th (ifn-γ) response to hrv infection is associated with increased viral shedding, and decreased proliferative response of pbmcs to hrv is associated with increased severity of symptoms. in addition, it was found that weak th responses (ifn-γ/il- mrna ratio) in sputum are also associated with greater severity of illness . furthermore, weak th responses to viral infection in adults with asthma have been associated with decreased lung function and greater airway responsiveness (brooks et al., ) . these results indicate that individuals with a weak th response to viruses, and perhaps individuals with asthma in general, may be more susceptible to hrv illnesses, and this association may be strongest in those with more severe disease (parry et al., ; gern et al., ; brooks et al., ) . other epidemiological and biological factors, such as allergy, atopic dermatitis, www.frontiersin.org or a family history of allergy, may be related to virus-induced asthma (green et al., ; singh et al., ) . recently it is suggested that variants at the q locus were associated with hrv induced asthma in children who had a history wheezing illnesses, although associations of q variants with asthma were restricted to children who had a history of hrv wheezing illnesses (calışkan et al., ) . human metapneumovirus is a recently identified rna virus belonging to the paramyxoviridae family, of genus metapneumovirus (collins and crowe, ) . hmpv is a major pathogen that causes ari in all ages (collins and crowe, ) . the first hmpv infection appears to take place within the first six months of life, after which infections may occur repeatedly and frequently (schildgen et al., ) . the nosocomial impact of hmpv is estimated to be as high as that for rsv. in an hmpv outbreak in japan, . % of elderly patients who shared the same day care room in a hospital were infected with hmpv (honda et al., ) . higher morbidity is observed in young children, the elderly, and immunocompromised adults (boivin et al., ; falsey et al., ; van den hoogen et al., ; sumino et al., ; williams et al., ; o'gorman et al., ) . hmpv is classified into two genotypes (a and b) and four subgroups (a , a , b , and b ) by phylogenetic analysis, using the f and g genes (biacchesi et al., ; van den hoogen et al., ) . subgroup a has been subdivided into two lineages, subgroup a a and a b (huck et al., ) . it has been suggested that these genotypes circulate in variable proportions in some areas (gerna et al., ; mackay et al., ) . although the molecular epidemiological information on hmpv has gradually accumulated, the detailed epidemiology remains unclear (mizuta et al., b; pitoiset et al., ; omura et al., ) . hmpv infections can occur throughout the year, but seasonality has been described in several studies, with the epidemiological peak occurring several months later than that observed for rsv epidemics (robinson et al., ; wilkesmann et al., ; madhi et al., ; aberle et al., aberle et al., , heininger et al., ). it remains unclear whether different hmpv subgroups are associated with differences in the clinical course of disease. several groups have suggested that hmpv subgroup a might be associated with more severe clinical disease (martinello et al., ; kaida et al., ; vicente et al., ; arnott et al., ) , while others have reported that subgroup b may cause more severe illness (esper et al., ; pitoiset et al., ) , and still other groups have found no evidence for differential severity caused by different hmpv lineages (agapov et al., ; manoha et al., ; larcher et al., ; xiao et al., ) . previous reports suggested that the substitution rates for the g gene ( . × − substitution/site/year) and the f gene ( . × − to . × − substitution/site/year) are high, and some positively selected sites have been found in the latter (de graaf et al., ; yang et al., ) . it may be that there is a correlation between some positively selected epitopes and disease severity. thus, the association between hmpv subgroup and disease severity is controversial. to gain a better understanding of host responses that may contribute to differences in clinical severity between hmpv subgroups, a more detailed analysis that includes host immunological status is needed. human parainfluenza virus belong to the paramyxoviridae family. there are two genera of hpiv, respirovirus (hpiv- and hpiv- ) and rubulavirus (hpiv- and hpiv- ; karron and collins, ) . hpiv is classified into four serotypes (hpiv - ) , all of which can cause various ari in humans such as uri, croup, bronchitis, asthma, and pneumonia (henrickson, ; karron and collins, ) . although hpiv type (hpiv ) is rarely reported, hpiv - are important causes of various ari, including the common cold, croup, bronchitis, bronchiolitis, and pneumonia in children, and they commonly re-infect both children and adults. while such infections are generally mild in healthy persons, they may cause serious diseases in children, such as asthma (henrickson, ; karron and collins, ) . although fewer hpiv strains have been detected compared with other respiratory viruses such as rsv, hrv, and hmpv, previous reports suggest that hpiv and are the dominant viruses in children with ari (reed et al., ) . indeed, serological surveys indicate that at least % of children have been infected with hpiv by years of age, approximately % have been infected by age , and at least % have been infected with hpiv by years of age (parrott et al., (parrott et al., , . hpiv and show high prevalence and are associated with up to % of acute lower respiratory tract infections in adults (azevedo et al., ; matsuse et al., ) . hpiv and hpiv , may be major agents of ari throughout the world, along with other viruses such as rsv, hrv, and hmpv (laurichesse et al., ; iwane et al., ; monto, ; do et al., ) . in addition, it is suggested that hpiv is a major causative agent of virus-induced asthma (henrickson and savatski, ) . several previous studies have reported that hpiv infections demonstrate clear outbreaks in autumn, mostly in september and november, every years (knott et al., ; hall, ; counihan et al., ) . other studies have reported that hpiv causes yearly outbreaks around the globe, mainly in the spring-summer season (knott et al., ; counihan et al., ; hall, ; mizuta et al., ) . a recent study suggested that four different types of hpiv cause similar clinical manifestations in patients, and the clinical presentation of hpiv infection may differ depending on patient age (liu et al., ) . henrickson and savatski ( ) analyzed the longitudinal evolution of the hn coding region in strains of hpiv isolated in the usa. these results showed that the antigenic and genetic subgroups are very stable. in addition, suggested that the evolution of the hn gene in the present hpiv isolates was relatively slow and that the gene is highly conserved. only a few reports on the molecular epidemiology of hpiv are available and it appears that the molecular epidemiology of hpiv is poorly understood. larger and more detailed studies on the association of hpiv with asthma are needed. hev was recently detected in asthmatic patients (hasegawa et al., ) . hev was found to be relatively acid resistant and thus could be distinguished from acid-sensitive hrv (schieble et al., ; kapikian et al., ) . hrv was recently reclassified frontiers in microbiology | virology as hev based on phylogenetic analysis and neutralization test, and some laboratories have confirmed its acid sensitivity ishiko et al., ; savolainen et al., ) . distinguishing between hrv and hev based on the acid sensitivity of isolates is therefore not appropriate for hev . the number of reports of an association between respiratory disease and hev infection has recently increased. one report of the phylogenetic analysis of hev based on partial vp gene sequences indicates wide genetic diversity (linsuwanon et al., ) . in addition, tokarz et al. ( ) showed the presence of multiple clades among the circulating strains, and that all strains are spreading rapidly worldwide and contributing to the prevalence rates of respiratory diseases. in addition, asthmatic individuals infected with hev also have the propensity to develop unstable asthma or an acute attack (hasegawa et al., ) . influenza virus is also a major causative agent of ari in both children and adults. furthermore, asthmatic patients were found among children and adults hospitalized with seasonal infv (dao et al., ; dawood et al., ) . although it is recognized that viral infections such as rsv or hrv may induce and/or exacerbate asthma, the effect of infv on asthma remains arguable (johnston et al., ) . although one study suggested that a(h n )pdm viruses impose greater risk factors on children than seasonal infv (tran et al., ) , infv vaccine was available before the influenza season since infv causes more severe illness than other respiratory viruses. therefore, it is suggested that infv vaccine be recommended for children with asthma (kloepfer et al., ) . although the level of detection of hcov, hbov, or adv is relatively low, these infections are also detected in children with acute wheezing (chung et al., ; jartti et al., ) . further studies are needed to clarify the clinical roles of hcov, hbov, or adv infections and those of other respiratory viruses. in particular, the prevalence of hcov, hbov, or adv infection in healthy control subjects, assessment of disease severity by other clinical variables, and the immunological effects should be investigated. infants with severe bronchiolitis have an increased risk of developing recurrent wheezing later in life (chung et al., ) . hrv may be detected concurrently with other viruses such as rsv, hmpv, infv, or hcov (richard et al., ; fujitsuka et al., ) . considering their ubiquity, it is interesting that the number of respiratory viruses detected concurrently with hrv strains is relatively low mackay, ) , supporting the concept that hrvs have a direct role in the clinical outcome of infection (miller et al., ) . in fact, hrv strains are co-detected with other pathogens in reproducible, but clinically undefined, patterns (brunstein et al., ) . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens while the host provides a vessel for hrv replication and transmission. respiratory viruses other than rsv and multiple viral infections may contribute to the severity of bronchiolitis and asthma. indeed, it was reported that dual infections of hmpv and rsv or hrv and rsv confer a -to -fold increase of severe disease in children admitted to pediatric intensive care units semple et al., ) . in contrast, other studies reported that co-infection with two respiratory viruses was not significantly associated with disease severity (van woensel et al., ; wolf et al., ) . thus, there is no consensus on the effects of co-infection on disease severity. the effect of dual infection may depend upon which viruses co-infect together. for example, although there was no increase in severity when hrv and/or adv were detected during rsv infection, co-infection with both hmpv and rsv increased the rate of intensive care unit admissions (aberle et al., ; semple et al., ) . thus, although dual infections and reinfections have been well documented in children, chronic infection with the development of quasispecies cannot be ruled out without obtaining more complete data using high performance detection methods (hall and mccarthy, ) . respiratory viral infections are a major cause of virus-induced asthma in early life. although antiviral therapy is not yet available for patients infected with respiratory viruses, the detection and identification of these viruses could help to explain serious respiratory illness, provide guidance for medical care, and prevent unnecessary treatment with antibiotics. based on the results of many related studies, we propose a two-step hypothesis of asthma development in children. the first step is mainly due to rsv infection: when rsv infects bronchial cells, the bronchial cells produce various cytokines and chemokines. these responses cause hyperresponsiveness in the bronchial cells. in other words, rsv infection might create a preparatory step as the first step in the development of asthma. hrv infection might then bring about the second step in the development of asthma. an infant with a history of wheezing caused by rsv infection may develop the heavy wheezing of asthma due to hrv infection followed by rsv infection. to understand the cause of asthma, we need to examine the larger complex picture of genetic susceptibility, immune components, environmental exposures, and the interactions between these elements. aberle single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response human metapneumovirus subgroup changes and seasonality during epidemics molecular epidemiological study of human rhinovirus species a, b and c from patients with acute respiratory illnesses in japan genetic variability of human metapneumovirus amongst an all ages population in cambodia between association of tlr polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children detection of influenza, parainfluenza, adenovirus and respiratory syncytial virus during asthma attacks in children older than years old genetic diversity between human metapneumovirus subgroups human rhinovirus and enterovirus represent a unique serotype with rhinovirus and enterovirus features virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups positive selection results in frequent reversible amino acid replacements in the g protein gene of human respiratory syncytial virus association of rhinovirus induced ifn-γ with increased asthma severity evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections role of viral respiratory infections in asthma and asthma exacerbations rhinovirus wheezing illness and genetic risk of childhood-onset asthma human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants molecular epidemiology of respiratory syncytial virus identification of variable domains of the attachment (g) protein of subgroup a respiratory syncytial viruses detection of viruses identified recently in children with acute wheezing respiratory syncytial virus and metapneumovirus human parainfluenza virus-associated hospitalizations among children less than five years of age in the united states. pediatr adult hospitalizations for laboratory-positive influenza during the burden of seasonal influenza hospitalization in children evolutionary dynamics of human and avian metapneumoviruses lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city economic impact of community-acquired and nosocomial lower respiratory tract infections in young children in germany genetic variability of human respiratory syncytial virus a strains circulating in ontario: a novel genotype with a nucleotide g gene duplication a -year experience with human metapneumovirus in children aged < years human metapneumovirus infections in young and elderly adults molecular epidemiology: focus on infection a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness nosocomial respiratory syncytial virus infections: prevention and control in bone marrow transplant patients the abcs of rhinoviruses, wheezing, and asthma relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons genotyping and phylogenetic analysis of major genes in respiratory syncytial virus isolated from infants with bronchiolitis in japan synergism between allergens and viruses and risk of hospital admission with asthma: case-control study il- gene expression in acute virus-induced asthma respiratory syncytial virus and parainfluenza virus respiratory syncytial virus," in mandell, douglas and bennett's principles and practice of infectious disease occurrence of groups a and b of respiratory syncytial virus over years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children enterovirus infection in children with asthma attacks: virus-induced asthma in japanese children human metapneumovirus infections -biannual epidemics and clinical findings in children in the region of basel, switzerland hospitalization for rsv bronchiolitis before months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study parainfluenza viruses two distinct human parainfluenza virus type genotypes detected during the milwaukee epidemic antigenic structure, function, and evolution of the hemagglutinin-neuraminidase protein of human parainfluenza virus type viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing outbreak of human metapneumovirus infection in elderly inpatients in japan novel human metapneumovirus sublineage pneumonia caused by rhinovirus human rhinovirus identified as human enterovirus by vp -based molecular diagnosis population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children wheezing rhinovirus illnesses in early life predict asthma development in high-risk children respiratory viruses and acute asthma in children incidence and characteristics of viral community-acquired pneumonia in adults the g glycoprotein of human respiratory syncytial viruses of subgroups a, and b: extensive sequence divergence between antigenically related proteins community study of role of viral infections in exacerbations of asthma in - year old children clinical response to antibiotic therapy for communityacquired pneumonia seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city a collaborative report: rhinoviruses -extension of the numbering system parainfluenza viruses," in fields virology different cytokine profile and eosinophil activation are involved in rhinovirus-and rs virus-induced acute exacerbation of childhood wheezing respiratory syncytial virus and human rhinoviruses are the major causes of severe lower respiratory tract infections in kuwait displacement of predominant respiratory syncytial virus genotypes in malaysia between genetic analysis of the vp /vp coding region in human rhinovirus species c in patients with acute respiratory infection in japan increased h n infection rate in children with asthma parainfluenza viral infections in pediatric outpatients: seasonal patterns and clinical characteristics causal links between rsv infection and asthma: no clear answers to an old question role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma molecular evolution of attachment glycoprotein (g) gene in human respiratory syncytial virus detected in japan community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parent-collected specimens masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during comparison of human metapneumovirus genotypes from the province of bolzano in northern italy with strains from surrounding regions in italy and austria epidemiological features of parainfluenza virus infections: laboratory surveillance in england and wales, - complete genome sequence of human respiratory syncytial virus genotype a with a -nucleotide duplication in the attachment protein g gene viral infections and asthma inception respiratory syncytial virus bronchiolitis high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease molecular epidemiology and evolution of human enterovirus serotype in thailand epidemiology and clinical presentation of the four human parainfluenza virus types human bocavirus; multisystem detection raises questions about infection genetic diversity of human metapneumovirus over consecutive years in australia seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodeficiency virus type- infection epidemiological and clinical features of hmpv, rsv and rvs infections in young children distinct patterns of evolution between respiratory syncytial virus subgroups a and b from new zealand isolates collected over thirty-seven years correlation between respiratory syncytial virus genotype and severity of illness naturally occurring parainfluenza virus infection in adults induces mild exacerbation of asthma associated with increased sputum concentrations of cysteinyl leukotrienes characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis analysis of genetic diversity and sites of recombination in human rhinovirus species c rhinovirusassociated hospitalizations in young children phylogenetic and cluster analysis of human rhinovirus species a (hrv-a) isolated from children with acute respiratory infections in yamagata endemicity of human metapneumovirus subgenogroups a and b in yamagata seasonal patterns of respiratory syncytial virus, influenza a virus, human metapneumovirus, and parainfluenza virus type infections on the basis of virus isolation data between detailed genetic analysis of hemagglutinin-neuraminidase glycoprotein gene in human parainfluenza virus type isolates from patients with acute respiratory infection between and in yamagata prefecture occurrence of respiratory virus: time, place and person quantitative and qualitative analysis of rhinovirus infection in bronchial tissues two distinct subtypes of human respiratory syncytial virus genotypic and phylogenetic analysis of the g gene of respiratory syncytial virus isolates in okinawa respiratory viruses and exacerbations of asthma in adults human metapneumovirus in adults: a short case series detection of human metapneumovirus genomes during an outbreak of bronchitis and pneumonia in a geriatric care home in shimane association of rhinovirus infection with increased disease severity in acute bronchiolitis rhinoviruses replicate effectively at lower airway temperatures acute respiratory diseases of viral etiology. iii. parainfluenza. myxoviruses clinical features of infection with hemadsorption viruses rhinovirusinduced pbmc responses and outcome of experimental infection in allergic subjects genetic variability in the g protein gene of group a and b respiratory syncytial viruses from india respiratory syncytial virus and metapneumovirus detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy human metapneumovirus genotypes and severity of disease in young children (n= ) during a -year study in dijon hospital, france whole genome sequencing and evolutionary analysis of human respiratory syncytial virus a and b from milwaukee epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children < years old the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis. pediatr seasonality and clinical features of human metapneumovirus infection in children in northern alberta premature stop codons in the g glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus a probable new human picornavirus associated with respiratory diseases human metapneumovirus: lessons learned over the first decade rhinovirus replication causes rantes production in primary bronchial epithelial cells dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis bronchiolitis-associated hospitalizations among us children asthma and allergy patterns over years after severe rsv bronchiolitis in the first year of life asthma and immunoglobulin e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age proposals for the classification of human rhinovirus species c into genotypically assigned types bronchiolitis to asthma: a review and call for studies of gene-virus interactions in asthma causation human rhinovirus infection in young african children with acute wheezing emerging respiratory agents: new viruses for old diseases? respiratory syncytial virus genetic and antigenic diversity detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment improved diagnosis of the etiology of communityacquired pneumonia with real-time polymerase chain reaction worldwide emergence of multiple clades of enterovirus comparison of children hospitalized with seasonal versus pandemic influenza a molecular epidemiology and disease severity of human respiratory syncytial virus in vietnam respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology genetic analysis of attachment glycoprotein (g) gene in new genotype on of human respiratory syncytial virus detected in japan rhinovirus," in fields virology novel respiratory syncytial virus subtype on among children antigenic and genetic variability of human metapneumoviruses prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection the epidemiology of respiratory syncytial virus (rsv) infections in south african children differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children severity of respiratory syncytial virus infection is related to virus strain neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections estimates of worldwide distribution of child deaths from acute respiratory infections human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults genetics, recombination and clinical features of human rhinovirus species c (hrv-c) infections; interactions of hrv-c with other respiratory viruses comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children the presence of rhinovirus in lower airways of patients with bronchial asthma prevalence and clinical and molecular characterization of human metapneumovirus in children with acute respiratory infection in china genetic diversity and evolution of human metapneumovirus fusion protein over twenty years severe bronchiolitis and respiratory syncytial virus among young children in hawaii molecular epidemiology of the attachment glycoprotein (g) gene in respiratory syncytial virus in children with acute respiratory infection in japan in / genetic variability of group a and b respiratory syncytial viruses isolated from provinces in china molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein this study was supported in part by research on emerging and re-emerging infectious diseases, labour, and welfare programs from the ministry of health, labour, and welfare, japan. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -cm vkpcn authors: fukuda, yosuke; akimoto, kaho; homma, tetsuya; baker, jonathan r; ito, kazuhiro; barnes, peter j; sagara, hironori title: virus-induced asthma exacerbations: sirt targeted approach date: - - journal: j clin med doi: . /jcm sha: doc_id: cord_uid: cm vkpcn the prevalence of asthma has increased worldwide. asthma exacerbations triggered by upper respiratory tract viral infections remain a major clinical problem and account for hospital admissions and time lost from work. virus-induced asthma exacerbations cause airway inflammation, resulting in worsening asthma and deterioration in the patients’ quality of life, which may require systemic corticosteroid therapy. despite recent advances in understanding the cellular and molecular mechanisms underlying asthma exacerbations, current therapeutic modalities are inadequate for complete prevention and treatment of these episodes. the pathological role of cellular senescence, especially that involving the silent information regulator homolog sirtuin (sirt) protein family, has recently been demonstrated in stable and exacerbated chronic respiratory disease states. this review discusses the role of sirt in the pathogenesis of bronchial asthma. it also discusses the role of sirt in inflammatory cells that play an important role in virus-induced asthma exacerbations. recent studies have hypothesized that sirt is one of major contributors to cellular senescence. sirt levels decrease in th and non-th -related airway inflammation, indicating the role of sirt in several endotypes and phenotypes of asthma. moreover, several models have demonstrated relationships between viral infection and sirt . therefore, targeting sirt is a novel strategy that may be effective for treating virus-induced asthma exacerbations in the future. asthma is the most common chronic respiratory disease, as over million individuals suffer from asthma worldwide [ ] . although the rate of asthma-related mortality has declined for decades due to the advancement in treatment strategies, the prevalence of asthma has gradually increased from to , and death rates have plateaued in some countries with aging populations [ ] . among all asthma patients, it is thought that about - % of patients have severe refractory asthma. this is due to inaccurate inhalation techniques, poor treatment adherence, and inadequate management of comorbidities [ ] . however, even when these factors are excluded, asthma is often still poorly controlled in the population. patients still have to grapple with various issues associated with the condition; asthma exacerbation is one of the challenges that requires a more effective solution. among multiple causes, infectious diseases are the most important cause of asthma exacerbations. various microorganisms, such as bacteria, fungi, and viruses, can cause acute exacerbations of asthma [ ] [ ] [ ] , and viral infection is the most common trigger. human rhinovirus (hrv) is one of the major pathogens of virus-induced asthma exacerbations. in a review of samples of hrv infection detected in the nasal mucosa of infants, hrv caused severe symptoms in winter, and hrv-a and hrv-c caused moderate to severe illnesses [ ] . a previous study confirmed elevated expression of interferon (ifn) and type cytokines analyzed from bronchosorption and nasosorption in asthma patients infected with hrv [ ] . in the presence of hrv infection, it was suggested that airway infections of streptococcus pneumoniae and moraxella catarrhalis might increase the risk of experiencing the severity and symptoms of asthma exacerbations [ ] . respiratory syncytial virus (rsv) is also an important pathogen for asthma exacerbations. rsv is known to infect almost all children by the age of years [ ] , and childhood rsv infection puts adults at risk of developing asthma [ ] . in addition, the annual incidence of rsv infection is - % in healthy older adults, and . % of patients hospitalized for asthma have comorbid rsv infection [ ] . thus, it is an essential pathogen for all generations. type and non-type airway inflammation are the two major immune phenotypes of asthma [ ] . these phenotypes and endotypes are defined by a variety of factors, including genetic predisposition and environmental factors such as antigen exposure, inflammatory biomarkers, tight junctions in the airway epithelium, and viral infections [ ] [ ] [ ] . in addition to the viral infections themselves, it has been suggested that viral infections can reduce the species, numbers, and diversity of microbiota, so-called dysbiosis [ ] , indicating that asthma is a very complex disease. viral infection, which can affect patients with both phenotypes, induces the formation of a wide range of cytokines and chemokines in the airway [ ] [ ] [ ] [ ] [ ] . eosinophils, t helper- (th ) lymphocytes, type innate lymphoid cells (ilc ), th cells, and neutrophils are involved in epithelial chemokine production in virus-induced asthma exacerbation [ ] [ ] [ ] [ ] [ ] (figure ). inhaled corticosteroids (ics), which have been used widely for treating asthma over the past few decades, inhibit the expression of inflammatory cytokines in the airway during virus-induced asthma exacerbations, especially th -airway inflammation [ ] . although ics ameliorate asthma exacerbation by limiting neutrophilic and non-th inflammation [ ] , it is also known that viral infection induces steroid resistance by inducing mainly neutrophilic airway inflammation [ ] . virus-induced asthma exacerbation overburdens healthcare systems, and it elevates the rates of morbidity and mortality [ ] . moreover, a few patients, described as severe, do not respond to current therapies, and few prophylactic strategies are available for treating such refractory cases. therefore, new therapeutic targets and approaches are the need of the hour. cellular senescence is characterized by irreversible cell-cycle arrest and release of inflammatory mediators known as the senescence-associated secreted phenotype (sasp), which can exert paracrine and autocrine effects on naïve cells [ ] . the silent information regulator homolog (sirt ), which is a nicotinamide adenine dinucleotide (nad+)-dependent class iii deacetylase, is one of the essential proteins that regulates aging, metabolism, dna repair, immunity, and inflammation, and it protects against cellular senescence [ , ] . in recent years, age-related diseases such as heart disease, neurological diseases, cancer, and diabetes have been found to be closely related to sirt [ ] , and similarly, sirt has garnered considerable attention because of its role in the pathogenesis of asthma [ ] [ ] [ ] . an sirt -targeted treatment strategy may be effective in patients with virus-induced asthma exacerbation, who respond inadequately to existing therapies. in this review, we discuss the function of sirt in inflammatory cells that play a role in virus-induced asthma exacerbations and examine the possibility of using sirt as a target for treating virus-induced asthma exacerbations in the future. activation of sirt induces sasp in t cells through deacetylation of several transcription factors, such as p , nf-κb, forkhead box o (foxo) , pi k, hif- α, and pgc α [ , ] , regulating autophagy, dna repair, mitochondrial function, and cellular senescence [ ] . it was suggested that the mechanism for this was an enhanced glycolysis in helper t cells, which might lead to immune dysfunction [ ] . on the other hand, the details of b cells involved in the humoral immune response are not yet well known [ ] . in , a screen for mammalian sirt activators identified sirt activators, including resveratrol, piceatannol, and quercetin, called sirtuin activating compounds (stacs). among them, resveratrol was shown to be the most potent activator of sirt [ ] . in a clinical trial of sirt activators in mild to moderate ulcerative colitis, srt , a sirt activator, was well tolerated [ ] . adverse events of srt was reported to include upper abdominal pain, fatigue, photophobia, diarrhea, and headache [ ] . it is considered to be a relatively safe drug to use. on the other hand, selisistat (ex ), a sirt inhibitor, has been studied in healthy individuals and patients with huntington's disease, and has also been shown to be safe [ , ] . the association between sirt inhibitors and respiratory illness is not well reported. based on these previous reports, we will discuss about the impact of sirt on virus-induced asthma exacerbations below. neutrophilic inflammation occurs in the airway during virus-induced asthma exacerbation. cxcl is a crucial cytokine of the neutrophilic airway inflammatory process, which is also involved in virus-induced asthma exacerbation [ ] . hrv infection increases cxcl and il- β levels in the nasal lavage fluid of patients with asthma [ ] . smoking is an important factor in the worsening of the disease in asthmatic patients. stimulating human airway epithelial cells with hrv infection and cigarette smoking extract (cse) enhances cxcl expression [ ] . increased production of cxcl is one of the mechanisms of corticosteroid resistance [ , ] . the antimicrobial drug azithromycin may be effective in asthmatics with a predominance of cxcl- and other neutrophilic cytokines [ , ] . in a clinical trial of children with rsv-infected bronchitis, azithromycin significantly reduced the expression of cxcl in nasal lavage and reduced respiratory symptoms one-year post-use compared to a placebo [ ] . azithromycin may exert its effects by inducing ifn-β and ifn / , but the clinical benefits of using antimicrobials, including azithromycin, in patients with asthma are still unclear [ , ] . mmp- , a type of matrix metalloprotease, has been found to be increased during viral infections [ ] . mmp- also causes airway remodeling through neutrophilic airway inflammation [ , ] . airway remodeling is associated with decreased respiratory function and disease severity [ ] . despite the fact that both cxcl and mmp- are important factors associated with neutrophilic airway inflammation, there are few therapeutic agents for these factors involved in neutrophilic airway inflammation. furthermore, it may lead to severe virus-induced asthma, and effective treatment strategies are needed. several reports suggest that sirt regulates neutrophilic airway inflammation related to cxcl [ ] [ ] [ ] [ ] . cse-induced cxcl elevation in mature mononuclear cells was attenuated by the overexpression of sirt in vitro [ ] . these studies showed that activation of nf-κb signaling and deacetylation of foxo a protein as mechanisms by which sirt regulates neutrophilic airway inflammation [ ] . a previous study confirmed that foxo a expression was upregulated by rsv infection [ ] . thus, sirt activators, including resveratrol, may be effective in targeting cxcl -induced neutrophilic airway inflammation in virus-induced and steroid-resistant asthma exacerbations [ , ] . interestingly, in basic experiments with macrophages isolated from bronchoalveolar lavages of copd patients, resveratrol inhibited the release of nearly all cytokines from alveolar macrophages. in contrast, dexamethasone, a type of systemic corticosteroid commonly used in the treatment of asthma exacerbations, only partially inhibited the release of cxcl [ ] . these lines of evidence suggest that activation of sirt may lead to suppression of neutrophilic inflammation, possibly through suppression of cxcl and may be an effective therapeutic strategy, especially for steroid-resistant virus-induced asthma exacerbations. suzuki et al. investigated the relationship between viral infections and mmp- expression using human nasal epithelial cells [ ] . notably, they found that the expression of mmp- , which was enhanced by poly(i:c), was attenuated by resveratrol. furthermore, in the presence of the sirt inhibitor splitomicin, poly(i:c) significantly enhanced the expression of mmp- [ ] . another study showed that the increased mmp- was attenuated by not only the sirt activator resveratrol, but also by the diabetes drug metformin in a mouse model exposed to uv light [ ] . these results indicated that sirt activation could be a novel therapeutic strategy for virus-induced asthma exacerbations by regulating mmp- expression and suppressing airway neutrophilic inflammation and remodeling. however, additional studies will be necessary to determine whether mmp- is a good biomarker for sirt -targeted therapy. eosinophils play an essential role in virus-induced asthma exacerbation. ccl and ccl , which are chemokines associated with eosinophils, may be upregulated and recruit eosinophils when a virus infects the airway epithelium [ ] . eosinophil cationic protein (ecp), an inflammatory mediator, is released by eosinophils and correlates with airway hyperreactivity [ ] . in addition to these, cytokines such as interleukin (il)- , il- , and il- are thought to be involved in a complex. calhoun et al. investigated whether hrv could trigger an allergic response in the airway [ ] since it is a significant viral pathogen that exacerbates asthma in adults and children [ ] . bronchoalveolar lavage was performed for both healthy and allergic participants with or without hrv infection, and they concluded that eosinophil recruitment in the airway occurred during or after hrv infection in allergic participants, but not in healthy participants [ ] . kato et al. studied childhood asthma and reported that serum il- and ecp levels were significantly higher in the virus-induced asthma group than those in the control group [ ] . they also showed that the profile of those cytokines and chemokines differed with age [ ] . fractional exhaled nitric oxide is a good indicator of eosinophilic airway inflammation [ ] . bjerregaard and colleagues reported that feno in virus-induced asthma exacerbation was higher than in the follow-up period [ ] . they demonstrated that patients with higher feno levels had significantly shorter time to arrive at asthma exacerbation than those patients with lower feno [ ] . activation of toll-like receptor (tlr ), a virus receptor within the airway epithelial cells, led to the induction of eosinophil-attracting chemokines (ccl , eotaxin) in the cellular bases [ ] [ ] [ ] [ ] [ ] . these findings suggested that eosinophilic airway inflammation is an important aspect of virus-induced asthma exacerbation. several studies have demonstrated the importance of sirt in eosinophilic airway inflammation. wang et al., using an ovalbumin-induced asthma mouse model, found that sirt was associated with eosinophilic airway inflammation [ ] . while they reported that serum sirt levels were increased in ova-sensitized and challenged mice model, sirt levels were decreased in lung tissue, and more il- , il- , and il- in balf were found in the ovalbumin-induced asthma mouse model compared to the controls [ ] . they also showed that respiratory function (forced expiratory volume in s/forced vital capacity) was negatively correlated with serum sirt in asthmatic human samples [ ] . based on these results, they believed that the elevated serum sirt levels were due to the release of sirt from the tissues following airway inflammation. in another study, sirt activator (sirt ) treatment decreased the eosinophil count and il- and il- levels, but not il- levels in the bronchoalveolar fluid and lung tissue in the ovalbumin-induced asthma mouse model [ ] . they also reported that sirt activation significantly inhibited inflammatory cell infiltration in the airways, but it did not significantly affect goblet cell hyperplasia, and they attributed this to the possibility that sirt activation might be inadequate to control airway inflammation. resveratrol, a known sirt activator, also attenuated il- and il- as well as eosinophil accumulation in ovalbumin-induced allergic rhinitis in mice [ ] . this may be attributed to the differential effect of sirt on transcription factors, including gata , which is a transcriptional factor that regulates th differentiation and the expression of the t cytokines il- , il- , and il- [ ] . sirt is a key regulator of gata via its deacetylation, and in t-lymphocytes from patients with severe asthma, a decrease in sirt has been linked to increased expression of il- via increased gata activation [ ] . these findings supported the fact that sirt activation might suppress eosinophilic inflammation in the airway during acute asthma exacerbations. controlling eosinophilic inflammation is a key approach for predicting and treating virus-induced asthma exacerbations [ , , ] . anti-il- therapies, such as mepolizumab and benralizumab, are effective against eosinophilic airway inflammation and markedly reduce virus-induced asthma exacerbations [ , ] . although these biologics produce marked effects in patients with refractory asthma, their efficacy was limited in patients with non-th -asthma [ , ] . activation of sirt may facilitate control of eosinophilic inflammation and refractory eosinophilic asthma. ige is an important therapeutic target for virus-induced asthma exacerbation, which is mainly produced by plasma cells. the position of omalizumab, which is a humanized anti-ige monoclonal antibody, has been confirmed as an important therapeutic agent [ ] . the prose study revealed a lower asthma exacerbation frequency in the omalizumab group than that in the placebo group [ ] . in this study, they conducted a subgroup analysis of patients with hrv infection and found a significant increase in ifn-α in the group of patients treated with omalizumab, which may be a protective mechanism for viral-induced asthma exacerbations [ ] . another clinical study showed that omalizumab decreased the frequency and duration of hrv infection in patients with childhood asthma [ ] . despite the efficacy of omalizumab in patients with asthma with viral infection, it was reported that some patients, especially geriatric patients, responded poorly to anti-ige. this observation may be attributed to immunosenescence, which includes impaired mucociliary clearance, changes within the inflammatory cells in the airway, and decreased antigen response [ ] . hence, there is a demand for other treatment options besides anti-ige therapy for patients who are unresponsive to anti-ige therapy. lipid profiling is thought to be important for understanding viral infections [ , ] . when the virus reacts with airway epithelium, mast cells are activated in an ige-dependent or independent manner, and degranulation occurs [ ] . this results in the production of lipid mediators such as prostaglandin (pg) and cysteinyl leukotriene (cyslts), which induce an immediate response in the airways and other target organs [ ] . currently, drugs targeting lipid mediators in asthma are mainly the cyslt receptor antagonists pranlukast and montelukast [ , ] . it is known that obesity, a factor in refractory asthma, results in steroid resistance due to decreased adipokines. although leukotriene receptor antagonists are useful in such patients [ ] , they are still not well controlled. a better understanding of the pathogenesis of refractory asthma by further approaches to lipid mediators is an important issue. resveratrol, a polyphenol found in grapes, berries, red wine, and peanuts [ ] , can activate sirt [ ] . lee et al. examined the possible anti-inflammatory effects of resveratrol in an ovalbumin-induced asthma mouse model [ ] . resveratrol significantly reduced total ige and ovalbumin-specific ige levels and increased serum igg a, which is associated with th response, in serum [ ] . moreover, it reduced airway hyperresponsiveness and mucus hypersecretion compared to a placebo [ ] . yet another study found that sirt regulated the pathways, amp-activated protein kinase (ampk), and protein tyrosine phosphatase b. modulation of these pathways via resveratrol attenuated signals from the ige receptor, fcεri, and inhibited the release of lipid mediators, leukotriene c (ltc ) and pgd , and the inflammatory cytokines, tumor necrosis factor (tnf)-α and il- [ ] . in a mouse model of ova-induced allergic rhinitis, sirt administration reduced symptoms such as sneezing and nasal rubbing events, and it led to a significant reduction in serum ige [ ] . it is known that when ige antibodies bind to the antigen, intracellular secretory granules are transported to the cell surface, and chemicals, such as histamine, contained in the granules are released. previous reports demonstrated that sirt inhibited degranulation [ ] . the study noted that inhibition of degranulation by sirt may be mediated through inhibition of the response mechanisms of the phosphorylation of protein kinase c (pkc) isomer, pkcµ and pkcθ. these data suggested that sirt activation may ameliorate ige-mediated airway inflammation in viral-induced asthma exacerbations, whereas the detailed mechanism by which omalizumab blocks ige is unclear and requires further study. some studies on the association between sirt and lipid mediators have been reported. tan et al. demonstrated in basic experiments using eosinophils isolated from whole human blood that trans-resveratrol suppressed the expression of ltc [ ] . another study showed that resveratrol reduced the expression levels of ltc and pgd in a mouse model of eosinophilic sinusitis [ ] . these results might be attributed to inhibition of the phospholipasea (pla ) and lipoxygenase (lox) pathways, which play an important role in the arachidonic acid cascade [ , ] . as mentioned above, sirt is closely related to metabolism. it was reported that fisetin and licochalcone, which are polyphenols, improve hepatic lipid metabolism via the sirt /ampk pathway in a mouse model [ , ] . in a clinical trial on asthma and diet, a healthier diet correlated with better asthma control [ ] . these evidences suggest that drug treatment and diet modification may be one of the lipid mediator-mediated sirt targeted treatment strategies for virus-induced asthma exacerbations. ilcs are a novel type of lymphocyte, which have been recently identified as playing an important role in immune diseases. th -type cytokines were initially found to be produced solely by th cells, but recent findings show that ilc cells, although less numerous than th cells, are more efficient in producing th -type cytokines [ ] . moreover, ilc has been shown to play an essential role in virus-induced asthma [ , ] . studies have confirmed that the expression of the upstream cytokines il- , il- , and tslp that regulate ilc is enhanced in rhinovirus infections [ ] [ ] [ ] [ ] . current knowledge suggests that airway epithelial damage triggered by viral infections promotes the production of il- , il- , and thymic stromal lymphopoietin (tslp), which in turn leads to the activation of ilc and exacerbation of asthma [ ] . we showed that the late addition of budesonide attenuates the increase in tslp caused by viral infection [ ] . although tezepermab, a biologic that targets tslp for treatment, was reported to reduce the frequency of asthma exacerbations [ ] , the role of ilcs , including il and il- , is not fully understood, and we need to elucidate the full mechanism of its production and develop new treatment options. little is currently known about the association between airway inflammation, sirt , and ilc . basic experiments using mouse models of allergic diseases demonstrated that resveratrol inhibited the expression of il- , il- , and tslp in airway epithelial cells [ ] . resveratrol also reduces caspase- , an indicator of apoptosis [ ] . in basic experiments using a mouse model of hdm-induced asthma, resveratrol reduced cell apoptosis and suppressed the expression of the γh ax gene, which is associated with dna damage [ ] . activation of sirt suppresses epithelial damage, which may inhibit apoptosis and control viral-induced asthma exacerbations. leptin is one of the adipokines secreted by adipocytes and is involved in increased energy metabolism and appetite suppression via hypothalamic receptors [ ] . an increase in sirt led to an increase in the sensitivity of leptin [ ] . it was suggested that elevated leptin could cause exacerbation of allergic diseases via ilc [ , ] . zeng et al. tested the relationship between leptin and ilc in patients with allergic rhinitis [ ] . the results showed that leptin expression correlated with the percentage of ilc in peripheral blood mononuclear cells. mapk signaling and pi k signaling were thought to be the possible pathways involved in this response [ , ] . it was reported that sirt was inhibited by p mapk and pi k signaling via micro-rna (mirna) -a and mirna expression, if the airway epithelial cells were subjected to oxidative stress [ , ] . the possible mechanisms by which sirt regulates allergic airway inflammation through ilc , such as cellular apoptosis and lipid metabolic pathways, as well as the presence of mirnas, require further investigation. th cells also play an important part in virus-induced asthma, mediated by the role of il- family cytokines [ ] [ ] [ ] . in basic experiments in which an ova mouse model was infected with rsv, il- a regulated the airway hyperreactivity [ ] . niwa et al. demonstrated in vitro experiments using normal human bronchial epithelium that the increase in ifn-λ, which plays a protective role in viral infection, was attenuated by the presence of il- a [ ] . increased il- is thought to be one of the mechanisms of steroid resistance in asthmatic patients [ ] , and il- may be a novel therapeutic target for patients with viral-induced asthma who are refractory to treatment. il- and transforming growth factor (tgf)-β are required for th differentiation. viral infection, including hrv infection, increases il- levels in the respiratory tract [ , ] , which correlates with airway remodeling and the severity of asthma [ , ] . at present, the anti-il- antibody tocilizumab, which is used to treat rheumatoid arthritis, is not indicated for asthma, but basic experiments suggested that il- may be an important marker of asthma [ , ] . tgf-β is an essential factor affecting airway remodeling along with mmp- , amphiregulin, vascular endothelial growth factor (vegf), and fibroblast growth factor (fgf) [ ] . repeated rv infection in mice not sensitized to allergens activated tgf-β in lung tissue, but neutralizing tgf-β reduced airway smooth muscle thickening [ ] . tgf-β-deficient mice showed an earlier increase in ifn-β in lung tissue compared to that in controls [ ] . collectively, tgf-β was found to contribute strongly to remodeling during virus-induced asthma exacerbations. one therapeutic candidate targeting th cells is brodalumab, a monoclonal antibody against the il- receptor, which has failed to show efficacy in the clinical trial [ ] . further elucidation of their mechanisms and development of therapeutic agents are required to control the pathogenesis of virus-induced asthma exacerbations. it was reported that loss of sirt further induced mrna expression of il- in an animal model of rsv infection [ ] . according to their considerations, sirt -deficient bone marrow dendritic cells elevated acetyl coa carboxylase (acc ), which is associated with fatty acid synthesis, resulting in the activation of an abnormal metabolic process, which in turn preceded an excessive virus-induced immune response [ ] . in other words, sirt may regulate the th immune response from virus-infected dendritic cells by regulating their metabolic pathways [ ] . the relationship between sirt and il- has been well studied in other diseases. previous studies investigated the effect of sirt activation in patients with psoriasis [ , ] . they found a significant histological improvement in the sirt activator group compared to that in the placebo group, which was attributed to the inhibition of il- and tnf-α [ ] . moreover, resveratrol, a sirt activator, suppressed the expression of ccl , a chemokine that is essential chemokine for the production of il- [ ] . the involvement of th cells was demonstrated in patients with diabetic ophthalmopathy [ , ] . other studies reported that the sirt activator might inhibit the elevation in serum il- levels, and regulation of il- through sirt probably leads to a reduction in the development of diabetic ophthalmopathy [ ] . by applying the proven relationship between sirt and il- in these other diseases to viral-induced asthma, sirt could become a new therapeutic target in the future. sirt regulated il- expression in the ovalbumin-induced asthma mouse model [ , , ] . these studies confirmed that the activation of the pi k-akt pathway led to an increase in il- , and this response was attenuated by sirt inhibitors [ , ] . ichikawa et al. showed that a sirt activator suppressed il- and tnf-α production by splenocytes in ovalbumin-challenged mice [ ] . these indicate that the akt-sirt signaling is a crucial pathway to the control of il- . interestingly, another study reported that metformin, a pharmacotherapeutic agent used to treat diabetes, reduced il- , il- , il- β, and tnf-α levels in a mouse model of acute respiratory distress syndrome [ ] . one possibility was that the low expression of mirna might suppress the mitogen-activated protein kinase (mapk) pathway upstream of sirt [ ] . moreover, metformin, like resveratrol, was reported to inhibit hif- α expression, but through a different pathway [ ] . in clinical practice, oral metformin reduced asthma-related hospitalizations and asthma exacerbations [ ] . metformin may be one of the treatment options in patients with virus-induced asthma exacerbations by regulating th cells. the relationship between sirt and tgf-β has often been studied in idiopathic pulmonary fibrosis (ipf). zeng et al. used a bleomycin-induced mouse model to explore the role of sirt in pulmonary fibrosis [ ] . they demonstrated that activation of sirt by resveratrol and srt inhibited myofibroblast differentiation induced by tgf-β . overexpression of sirt , a member of the sirt family, reduced e-cadherin, a marker of emt, through the tgf-β pathway [ ] . this result might involve p , a protein that regulates cell cycle progression [ ] . another study showed that resveratrol inhibits airway remodeling through transforming growth factor (tgf)-β /smad signaling [ ] . the relationship between sirt and tgf-β, which was identified in ipf, has potential applications in virus-induced asthma exacerbations, but the mechanisms of this relationship are still unclear. therefore, the findings of these studies suggest that future treatments against cellular senescence, especially sirt , may regulate th airway inflammation. we believe that metformin is an attractive treatment option because it is already used in many patients with few adverse events, but further research is needed to determine whether it is useful in patients with virus-induced asthma exacerbations who do not have diabetes mellitus. protein acetylation plays a crucial role in host response to viral infection. histone deacetylases (hdac) are enzymes that define chromatin structure and are closely associated with chronic respiratory diseases [ ] . adenovirus infection resulted in reduced activation of hdac in ova sensitization mice [ ] . experiments using human blood samples also showed reduced activation of hdac in asthmatics compared to that in healthy subjects [ ] . nf-e -related factor (nrf ) is a transcription factor that has a protective effect on cells from oxidative stress caused by reactive oxygen species. rsv, which frequently causes asthma exacerbations, induces deacetylation of nrf [ ] . in vitro models of rhinovirus infection showed that inhibition of s-nitrosoglutathione reductase was reported to increase sqstm , an nrf -dependent gene, and suppress viral growth, with an effect on airway hypersensitivity [ ] . although these viral proteins are important factors in the pathogenesis of virus-induced asthma exacerbations, there remain unanswered questions concerning established treatment. theophylline, a drug for asthma and copd, activates hdac and exerts an anti-inflammatory effect. when the activity of hdac was investigated in lps-stimulated macrophages, hdacs were activated by the combination of theophylline compared to dexamethasone alone [ ] . in a clinical trial examining the effects of low volume theophylline, the addition of theophylline to inhaled steroids improved respiratory function [ ] . in patients with acute exacerbations of copd, theophylline also improved hdac activity during the stable phase [ ] . interestingly, steroid resistance correlated with a decrease in sirt, and a combination of steroid, theophylline, curcumin, or resveratrol treatment resulted in an increase in sirt as well as an increase in glucocorticoid receptors [ ] . doxophylline, which is considered to have fewer side effects than conventional theophylline preparations, improved the protein expression of sirt , which was reduced by lps stimulation [ ] . as a treatment option for targeting sirt , theophylline is very effective for patients with inadequate response to steroids. sirt regulates the expression of various genes by deacetylating histones and transcription factors, such as nf-kb, stat , and stat [ ] [ ] [ ] [ ] . sirt deacetylates induced nf-kb activation in monocytes [ ] . sirt inhibits growth hormone-stimulated stat activation in mouse embryonic cells via the deacetylation of stat [ ] . treatment with sirt agonists deacetylates stat , which inhibits t-cell differentiation into th and th cells [ ] . a recent study reported that the activation of sirt by viral infection further affected stat activation [ ] . these studies indicated that transcription factors, including stat and stat , may be potential biomarkers in virus-induced asthma with sirt -targeted therapy. sirt also regulates antioxidant genes, which are important antiaging genes, via deacetylation of foxo and nrf . resveratrol increased the expression of nrf in obese rat models and paraquat-induced lung injury mouse models [ , ] . apios americana medikus is an edible tuberous legume native to eastern north america. chu and colleagues showed that apios americana medikus leaf extract increased the expression of nrf in mouse macrophages stimulated with lps [ ] . at present, there is no specific treatment for nrf , and further studies are needed. roflumilast, a selective inhibitor of phosphodiesterase (pde ), was reported to reduce exacerbations and hospitalization rates as a treatment for copd [ ] . roflumilast is highlighted by the fact that it increased the sirt expression along with nrf expression in copd patients [ ] . in vitro, rsv infection increased the nrf expression and its increase was attenuated by roflumilast [ ] . the proinflammatory cytokines il- , il- , and tnf-α were also reduced in a capacity-dependent manner. these results suggest that nrf may be a potential therapeutic target for viral asthma exacerbations. middle east respiratory syndrome coronavirus (mers-cov) pp ab protein is potentially regulated by sirt [ ] . sars-cov- might have a similar motif, but further study is required. sirt and other sirtuins were also reported to have antiviral roles against several dna and rna viruses, including hcmv, hsv- , adenovirus, and influenza a [ ] . although resveratrol was found to inhibit hrv replication in nasal epithelial cells [ ] , it is unclear whether this is due to intercellular adhesion molecule- (icam- ) as the hrv receptor, or reduction or deacetylation on potential viral protein acetylation, as the acetylation of hrv or rsv proteins remains unclear. caspase cleavage levels, indicators of apoptosis, are known to elevate in mers-cov infection [ ] , and resveratrol was found to reduce caspase cleavage levels with less cytotoxicity [ ] . activation of sirt may reduce apoptosis through deacetylation of viral proteins. in another report using airway epithelial cells, kim and colleagues examined the relationship between cellular senescence and the replication efficiency of influenza virus [ ] . senescent cells infected with influenza virus had reduced expression of ifn-β, which plays an essential role in the immune response compared to nonsenescent cells. they also examined whether sirt , an essential factor in cellular senescence, affected viral replication. interestingly, sirt -knockdown cells showed enhanced expression of proteins associated with influenza virus and reduced cell viability [ ] . collectively, with further research, therapies targeting sirt may control asthma exacerbations through acetylation of the viral protein. we reviewed the cellular interactions between sirt and inflammatory cells involved in virus-induced asthma exacerbations (table ). viral infection is a common health problem for children and adults with asthma. especially with the recent rampant covid- pandemic, treating and preventing viral infections is becoming an area of focus. while the role of various respiratory viruses in inducing the exacerbation of asthma is well established, the pathophysiological mechanisms underlying virus-induced asthma exacerbation and its treatment remain controversial. existing drugs such as ics and biologics are among the best treatment options for asthma exacerbation triggered by viral infection, but they may be partially ineffective due to unknown mechanisms. the current guidelines do not consider the potential for therapeutic agents related to sirt . the difference in the role of sirt in asthma between adults and children is not clear. however, as mentioned above, sirt -related signaling pathways are closely related to airway inflammation in asthma. a better understanding of the sirt mechanism may aid in the prevention and treatment of virus-induced asthma exacerbations ( figure ). the development of therapies targeting sirt could be a boon for patients with virus-induced asthma. further research is needed to clarify the relationship between known drugs and sirt and to explore the development of new drugs related to sirt . funding: this research received no external funding. regional, and national deaths, prevalence, disability-adjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, - : a systematic analysis for the global burden of disease study global asthma prevalence in adults: findings from the cross-sectional world health survey asthma in the elderly: what we know and what we have yet to know beneficial effect of early intervention with garenoxacin for bacterial infection-induced acute exacerbation of bronchial asthma and chronic obstructive pulmonary disease high burden of aspergillus fumigatus infection among chronic respiratory diseases rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th / cytokine and il- production human rhinovirus species and season of infection determine illness severity a comprehensive evaluation of nasal and bronchial cytokines and chemokines following experimental rhinovirus infection in allergic asthma: increased interferons (ifn-γ and ifn-λ) and type in fl ammation (il- and il- ) detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations hospitalization for rsv bronchiolitis before months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study respiratory morbidity years after rsv infection in infancy respiratory syncytial virus infection in elderly and high-risk adults mechanisms and management of asthma exacerbations precision medicine and phenotypes, endotypes, genotypes, regiotypes, and theratypes of allergic diseases tight junction, mucin, and inflammasome-related molecules are differentially expressed in eosinophilic, mixed, and neutrophilic experimental asthma in mice monitoring inflammatory heterogeneity with multiple biomarkers for multidimensional endotyping of asthma the role of lung and gut microbiota in the pathology of asthma cooperative activation of ccl expression by tlr and tumor necrosis factor-α or interferon-γ through nuclear factor-kb or stat- in airway epithelial cells inhibition of virus-induced cytokine production from airway epithelial cells by the late addition of budesonide proton pump inhibitors decrease eotaxin- /ccl expression in patients with chronic rhinosinusitis with nasal polyps: possible role of the nongastric h,k-atpase role of aspergillus fumigatus in triggering protease-activated receptor- in airway epithelial cells and skewing the cells toward a t-helper bias suppression of epithelial signal transducer and activator of transcription activation by extracts of aspergillus fumigatus basic research on virus-induced asthma exacerbation: inhibition of inflammatory chemokine expression by fluticasone propionate new molecular targets for the treatment of neutrophilic diseases models of respiratory infections: virus-induced asthma exacerbations and beyond europe pmc funders group europe pmc funders author manuscripts a complex secretory program orchestrated by the inflammasome controls paracrine senescence epstein lecture: sirtuins, aging, and medicine role of silent information regulator (sirt ) in regulating oxidative stress and inflammation. inflammation . ahead of print sirt as a therapeutic target in inflammaging of the pulmonary disease cellular senescence as a mechanism and target in chronic lung diseases metabolic reprogramming of human cd + memory t cells through loss of sirt resveratrol inhibits cd + t cell activation by enhancing the expression and activity of sirt sirtuins in b lymphocytes metabolism and function small molecule activators of sirtuins extend saccharomyces cerevisiae lifespan assessing colonic exposure, safety, and clinical activity of srt patients with mild to moderate ulcerative colitis pharmacogenomics and qt concentration-effect modelling of the sirt inhibitor selisistat in healthy volunteers an exploratory double-blind, randomized clinical trial with selisistat, a sirt inhibitor, in patients with huntington's disease. br asthma and natural colds inflammatory indices in induced sputum: a feasibility study interleukin- β and interleukin- ra levels in nasal lavages during experimental rhinovirus infection in asthmatic and non-asthmatic subjects cigarette smoke modulates rhinovirus-induced airway epithelial cell chemokine production steroid-resistant neutrophilic inflammation in a mouse model of an acute exacerbation of asthma novel approaches to the management of noneosinophilic asthma randomized trial to evaluate azithromycin's effects on serum and upper airway il- levels and recurrent wheezing in infants with respiratory syncytial virus bronchiolitis identification of novel macrolides with antibacterial, anti-inflammatory and type i and iii ifn-augmenting activity in airway epithelium effect of azithromycin on asthma exacerbations and quality of life in adults with persistent uncontrolled asthma (amazes): a randomised, double-blind, placebo-controlled trial azithromycin for acute exacerbations of asthma: the azalea randomized clinical trial human rhinovirus infection up-regulates mmp- production in airway epithelial cells via nf-{kappa}b sirtuin- controls poly (i:c)-dependent matrix metalloproteinase activation in primary human nasal epithelial cells activation of tgf-beta/smad signaling is associated with airway remodeling in asthma the effects of resveratrol on inflammation and oxidative stress in a rat model of chronic obstructive pulmonary disease sirt inhibits rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and inflammatory response via suppressing nf-kb pathway is decreased in lungs of patients with chronic obstructive pulmonary disease anti-inflammatory effects of resveratrol in lung epithelial cells: molecular mechanisms resveratrol delays postovulatory aging of mouse oocytes through activating mitophagy resveratrol attenuates the release of inflammatory cytokines from human bronchial smooth muscle cells exposed to lipoteichoic acid in chronic obstructive pulmonary disease sirt suppresses the senescence-associated secretory phenotype through epigenetic gene regulation resveratrol impairs the release of steroid-resistant cytokines from bacterial endotoxin-exposed alveolar macrophages in chronic obstructive pulmonary disease negative regulation of stress-induced matrix metalloproteinase- by sirt in skin tissue the role of rhinovirus in asthma exacerbations serum ecp and mpo, but not urinary lte , are associated with bronchial hyper-responsiveness a common cold virus, rhinovirus , potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects molecular epidemiology of respiratory viruses in virus-induced asthma differential effects of corticosteroids on serum eosinophil cationic protein and cytokine production in rhinovirus-and respiratory syncytial virus-induced acute exacerbation of childhood asthma allergology international virus detection and cytokine profile in relation to age among acute exacerbations of childhood asthma fractional exhaled nitric oxide as a predictor of response to inhaled corticosteroids in patients with non-specific respiratory symptoms and insignificant bronchodilator reversibility: a randomised controlled trial high fractional exhaled nitric oxide and sputum eosinophils are associated with an increased risk of future virus-induced exacerbations: a prospective cohort study peripheral sirt : a new biological characteristic of asthma sirtuin activator srt suppresses inflammation in an ovalbumin-induced mouse model of asthma resveratrol-mediated sirt activation attenuates ovalbumin-induced allergic rhinitis in mice defective sirtuin- increases il- expression through acetylation of gata- in patients with severe asthma asthma exacerbations and sputum eosinophil counts: a randomised controlled trial blood eosinophil count and prospective annual asthma disease burden: a uk cohort study anti-il- in mild asthma alters rhinovirus-induced macrophage, b-cell, and neutrophil responses (material) a placebo-controlled, double-blind study an early innate response underlies severe influenza-induced exacerbations of asthma in a novel steroid-insensitive and anti-il- -responsive mouse model cluster analysis and characterization of response to mepolizumab: a step closer to personalized medicine for patients with severe asthma baseline patient factor impact on the clinical efficacy of benralizumab for severe asthma enhanced plasmacytoid dendritic cell antiviral responses after omalizumab asthma and lower airway disease preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations effects of omalizumab on rhinovirus infections, illnesses, and exacerbations of asthma age-related changes in immune function: effect on airway inflammation that induce and resolve inflammation dysregulation of lipidomic profile and antiviral immunity in response to hyaluronan in patients with severe asthma current perspective on eicosanoids in asthma and allergic diseases-eaaci task force consensus report, part i. allergy . ahead of print effects of a short course of pranlukast combined with systemic corticosteroid on acute asthma exacerbation induced by upper respiratory tract infection montelukast reduces asthma exacerbations in -to -year-old children with intermittent asthma influence of body mass index on the response to asthma controller agents influence of resveratrol on the immune response resveratrol possesses protective effects in a pristane-induced lupus mouse model anti-inflammatory and anti-asthmatic effects of resveratrol, a polyphenolic stilbene, in a mouse model of allergic asthma sirt negatively regulates fc ri-mediated mast cell activation through ampk-and ptp b-dependent processes. sci. rep. sirt attenuates murine allergic rhinitis by downregulated hmgb /tlr pathway resveratrol inhibits ige-mediated basophilic mast cell degranulation and passive cutaneous anaphylaxis in mice trans-resveratrol, an extract of red wine, inhibits human eosinophil activation and degranulation resveratrol prevents development of eosinophilic rhinosinusitis with nasal polyps in a mouse model fisetin protects against hepatic steatosis through regulation of the sirt /ampk and fatty acid β-oxidation signaling pathway in high-fat diet-induced obese mice protective effects of licochalcone a ameliorates obesity and non-alcoholic fatty liver disease via promotion of the sirt- /ampk pathway in mice fed a high-fat diet associations between dietary scores with asthma symptoms and asthma control in adults pulmonary group innate lymphoid cells: surprises and challenges respiratory viral infection, epithelial cytokines, and innate lymphoid cells in asthma exacerbations t cells and ilc s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice the innate cytokines il- , il- , and tslp cooperate in the induction of type innate lymphoid cell exansion and mucous metaplasia in rhinovirus-infected immature mice il- -dependent type inflammation during rhinovirus-induced asthma exacerbations in vivo virus/allergen interactions in asthma thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type response to infection tezepelumab in adults with uncontrolled asthma resveratrol decreases cell apoptosis through inhibiting dna damage in bronchial epithelial cells age-associated weight gain, leptin, and sirt : a possible role for hypothalamic sirt in the prevention of weight gain and aging through modulation of leptin sensitivity leptin regulated ilc cell through the pi k/akt pathway in allergic rhinitis leptin enhances th and ilc responses in allergic airway disease microrna- is a novel regulator of cellular senescence and inflammaging resveratrol inhibits il- -mediated mast cell activation by targeting the mk / -pi k/akt axis il- a inhibits airway reactivity induced by respiratory syncytial virus infection during allergic airway inflammation nasal cytokine profiles of patients hospitalised with respiratory wheeze associated with rhinovirus il- a attenuates ifn-λ expression by inducing suppressor of cytokine signaling expression in airway epithelium decleyre-badiu, i.; dewilde, a. virus-triggered exacerbation in allergic asthmatic children: neutrophilic airway inflammation and alteration of virus sensors characterize a subgroup of patients oncostatin m: an interleukin- -like cytokine relevant to airway remodelling and the pathogenesis of asthma therapeutic potential of anti-il- therapies for granulocytic airway inflammation in asthma allergen-induced il- trans-signaling activates γδ t cells to promote type and type airway inflammation critical role of il- in dendritic cell-induced allergic inflammation of asthma tumor necrosis factor family member light acts with il- β and tgf-β to promote airway remodeling during rhinovirus infection epithelial-derived tgf-β acts as a pro-viral factor in the lung during influenza a infection randomized, double-blind, placebo-controlled study of brodalumab, a human anti-il- receptor monoclonal antibody, in moderate to severe asthma sirtuin regulates mitochondrial function and immune homeostasis in respiratory syncytial virus infected dendritic cells a randomized, placebo-controlled study of srt , a sirt activator resveratrol ameliorates imiquimod-induced psoriasis-like skin inflammation in mice prevention and treatment of diabetes with resveratrol in a non-obese mouse model of type diabetes protective effects of sirt in patients with proliferative diabetic retinopathy via the inhibition of il- expression effects of sirt /akt pathway on chronic inflammatory response and lung function in patients with asthma suppression of sirtuin- increases il- expression by activation of the akt pathway during allergic asthma metformin relieves acute respiratory distress syndrome by reducing mir- expression the role of metformin and resveratrol in the prevention of hypoxia-inducible factor α accumulation and fibrosis in hypoxic adipose tissue metformin use and asthma outcomes among patients with concurrent asthma and diabetes activation and overexpression of sirt attenuates lung fibrosis via p sirtuin attenuates epithelial-mesenchymal transition by suppressing the tgf-β /smad pathway and c-jun in asthma models accelerated epithelial cell senescence in ipf and the inhibitory role of sirt in tgf-β-induced senescence of human bronchial epithelial cells inhibitory effects of resveratrol on airway remodeling by transforming growth factor-β/smad signaling pathway in chronic asthma model decreased histone deacetylase activity in chronic obstructive pulmonary disease hdac -selective inhibitor rgfp demonstrates anti-inflammatory properties in raw . macrophages and mouse precision-cut lung slices by attenuating nf-κb p transcriptional activity expression and activity of histone deacetylases in human asthmatic airways respiratory syncytial virus infection down-regulates antioxidant enzyme expression by triggering deacetylation-proteasomal degradation of nrf . free radic histone acetylase and deacetylase activity in alveolar macrophages and blood mononocytes in asthma a molecular mechanism of action of theophylline: induction of histone deacetylase activity to decrease inflammatory gene expression effect of low-dose theophylline plus beclometasone on lung function in smokers with asthma: a pilot study low-dose theophylline enhances the anti-inflammatory effects of steroids during exacerbations of copd lymphocyte senescence in copd is associated with decreased sirtuin expression in steroid resistant pro-inflammatory lymphocytes the protective effect of doxofylline against lipopolysaccharides (lps)-induced activation of nlrp inflammasome is mediated by sirt in human pulmonary bronchial epithelial cells nad+-dependent sirt deacetylase participates in epigenetic reprogramming during endotoxin tolerance stat inhibition of gluconeogenesis is downregulated by sirt sirtuin- activation controls tumor growth by impeding th differentiation via stat deacetylation rhinovirus-induced sirt- via tlr regulates subsequent type i and type iii ifn responses in airway epithelial cells resveratrol protects against oxidative stress by activating the keap- /nrf antioxidant defense system in obese-asthmatic rats resveratrol protects mice from paraquat-induced lung injury: the important role of sirt and nrf antioxidant pathways flavonoids from apios americana medikus leaves protect raw . cells against inflammation via inhibition of mapks, akt-mtor pathways, and nfr activation effect of roflumilast on exacerbations in patients with severe chronic obstructive pulmonary disease uncontrolled by combination therapy (react): a multicentre randomised controlled trial the phosphodiesterase- inhibitor roflumilast reverts proteolysis in skeletal muscle cells of patients with copd cachexia roflumilast inhibits respiratory syncytial virus infection in human differentiated bronchial epithelial cells identification of lysine acetylation sites on mers-cov replicase pp ab sirtuins are evolutionarily conserved viral restriction factors resveratrol inhibits rhinovirus replication and expression of inflammatory mediators in nasal epithelia middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways effective inhibition of mers-cov infection by resveratrol enhanced viral replication by cellular replicative senescence this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we give thanks to manami matsuda and teru haba for their generous support. we thank editage (www.editage.jp) for english language editing. the authors declare no conflict of interest. kazuhiro ito is an employee of pulmocide ltd. key: cord- -fia w jt authors: ireland, d. c.; kent, j.; nicholson, k. g. title: improved detection of rhinoviruses in nasal and throat swabs by seminested rt‐pcr date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: fia w jt a seminested rt‐pcr (nrt‐pcr) was used to detect picornavirus (pv) rna in cell cultures inoculated with rhinoviruses (hrvs) and enteroviruses (evs). pcr tests in which a primary "touchdown" pcr was followed by secondary reactions using pv or hrv specific primers were able to differentiate hrvs of serotypes from evs. pvnrt‐pcr and hrvnrt‐pcr were then used to test nasal and throat swabs from adult subjects with naturally acquired respiratory virus infections. the swabs were also analysed for respiratory viruses by cell culture techniques and the rates of pv identification by the two methods were compared. pvnrt‐pcr was found to be at least five times more sensitive than cell culture for the detection of pvs in these clinical specimens. paired acute and convalescent serum samples were tested for complement fixing antibodies to adenovirus, influenza a and b, respiratory syncytial virus, parainfluenza viruses , , and , myco plasma pneumoniae, and chlamydia psittaci. an enzyme‐linked immunosorbent assay (elisa) was used to detect rises in antibody level to coronavirus types e and oc . the overall rate of pathogen identification in swabs from adult asthmatics increased from % when only cell culture and serology were used to % when these methods were supplemented by pvnrt‐pcr. © wiley‐liss, inc. the common cold is a major cause of morbidity, loss of work, and drug expenditure. each year the average preschool child has - colds and the average adult - colds [sperber and hayden, . research into the effects of colds on normal individuals and on those with chronic lung disease has been hampered by the considerable dieculties encountered in identifying respiratory pathogens. human rhinoviruses (hrvs), the major causative agents of the common cold [couch, , are ogy for screening [hamparian et al., . rates of virus identification have rarely been greater than %. minor and colleagues ( ) recovered hrvs from % of specimens from young asthmatic children with symptomatic respiratory infections, but were only able to isolate hrvs from % of specimens from older patients [minor et al., . the rates of total virus identification in these studies, using a variety of diagnostic methods, were and %. nucleic acid hybridization techniques, particularly those based on the polymerase chain reaction [saiki et al., , are now being used for rapid detection of viruses and slow-growing organisms [hayden et al., . several short conserved regions in the ' untranslated sections of picornavirus (pv) genomes [duechler et al., ; hughes et al., have provided sequences for oligonucleotide probes and primers gama et al., gama et al., , hyypia et al., ; torgersen et al., . four of these regions (a-d) are shown in fig. . radiolabelled synthetic oligonucleotide probes ( mers) complementary to c and d were used by bruce and colleagues [ ] to detect the rna of hrv serotypes and to identify hrv infection in nasal washes from experimentally infected volunteers. the probes gave results comparable to conventional virus culture on days two and three post-inoculation, but were more sensitive than culture on subsequent days. negative results were obtained with unrelated respiratory viruses including reoviruses, influenza viruses, coronavirus and herpes simplex virus. oligonucleotides ( - mers) complementary to c and d were used as reverse transcriptase and backward pcr primers by several groups of workers [gama et al., [gama et al., , hyypia et al., ; torgersen et al., . all groups used part of the conserved a sequence ( or mers) for the forward pcr primer. gama and colleagues [ ] obtained positive results for nasal washings from all volunteers experimentally infected with hrv , hrv or hrv . the reaction products were versity. all subjects were adults: two groups were nor-ma healthy students and the third group was asthmatic. ethical committee amroval and signed informed .* u consent were obtained. fig. . four conserved regions in the '-untranslated regions of rhinoviruses that have provided sequences for oligonucleotide probes and rt-pcr primers. numbering shown is for hrvl [stanway et al., . analysed by agarose gel electrophoresis after reaction cycles and further characterised by nucleotide sequencing. the complementary sequence to region b ( mer) was used by hyypia et al. [ as an hrv probe for southern blots, who reported results for wild-type pvs isolated from clinical material in llc or hela cells. all except one gave an amplification product of the expected size but only out of products from hrvs hybridized with the probe. olive et al. [ ] used sequence d ( mer) for a forward pcr primer with a reverse transcriptase and backward pcr primer ( mer) complementary to a sequence in the downstream vp -coding region. the reaction products were identified solely by their size as seen in ethidium bromide-stained agarose gels. hrvs could be differentiated from other pvs by this method: pcr products were bp for hrvs and bp for polioviruses, coxsackieviruses, and echoviruses. these workers used pcr directly on nasal swabs but confined their efforts to specimens which had been positive for respiratory viruses by cell culture. we have devised seminested rt-pcrs (nrt-pcrs) to improve the sensitivity and specificity of this method of pv detection. the "pcr uses a primer of sequence a and a primer complementary to sequence d in a "touchdown" reaction cycle to reduce spurious priming [don et al., . "pcrs using a primer of sequence a together with primers complementary to either b (hrvnrt-pcr) or c (pvnrt-pcr) are then used to differentiate between hrvs and other pvs. we have applied this test directly to nasal and throat swabs from controls and from individuals with naturally acquired colds and compared the rate of pv detection by pvnrt-pcr and by cell culture. clinical samples were taken from volunteers in two studies at leicester university and one at cardiff uni- subjects in leicester were asked to record symptoms on a -day diary card (as described previously, wiselka et al., ) . symptoms were use of handkerchiefs, runny nose, stuffy nose, sneezing, sore throat, hoarseness, red or watery eyes, face-, head-, or earache, malaise, muscle aches, chills, cough, and painful, swollen neck glands. symptomatic days were those on which two or more symptoms were recorded with at least one symptom moderately severe or worse. the number of symptomatic days and the total symptom score for the ten days were used as a measure of the severity of the cold and those subjects with doubtful colds were excluded from the study group. volunteers in cardiff were self-diagnosed as suffering from colds. they were not selected for this study on the basis of their symptom scores. nasal swabs were placed high in the anterior nares and throat swabs were passed firmly over the pharynx and tonsils. swabs were placed together in . ml of virus transport medium containing nutrient broth ( % foetal calf serum, penicillin, streptomycin, and amphotericin b). all specimens were then stored at volumes ( . ml) of nasal and throat swabs were inoculated onto monolayers of ohio hela cells, mrc- human lung fibroblasts, c cells (a cell line derived from mrc- fibroblasts, susceptible to coronavirus), and madin-darby canine kidney (mdck) cells. all cell lines were cultured in roller tubes at °c with % coz and observed for days. specimens inoculated onto ohio hela cells were routinely passaged once after days and equivocal specimens were passaged up to three times. hrv infection was diagnosed after observation of characteristic cytopathic effect (cpe). positive isolations of hrv were confirmed by demonstrating characteristic acid lability at ph and inhibition of cpe at °c. influenza viruses were identified by haemadsorption inhibition on mdck cells. pvs of known serotype obtained from the mrc common cold unit, salisbury, u.k. were also cultured. echoviruses and coxsackie viruses were grown in mrc- cells and hrvs in hela cells. paired acute and convalescent sera were tested for complement fixing antibodies to adenovirus, influenza a and b, respiratory syncytial virus, parainfluenza viruses , , and , mycoplasma pneumoniae, and chlamydia psittaci. an exzyme-linked immunosorbent assay (elisa) [kraaijeveld et al., was used to detect rises in antibody level to coronavirus and a similar elisa, using antigen prepared from infected suckling mouse brain, was used to detect antibodies to coronavirus oc . - °c. samples ( pl) of cell culture grown virus or nasal and throat swabs in transport medium were used for nucleic acid extraction. this material was transferred immediately on thawing to . ml reaction tubes containing pg proteinase k and pg carrier trna in p x proteinase k buffer ( mm tris-hc , ph . , mm edta, % sds). appropriate negative controls were always included. after incubation at °c for min, the samples were extracted once with phenol/ chloroform and twice with chloroform. nucleic acid was precipitated by the addition of pl of m sodium acetate, ph . , and ml of cold ethanol to each tube. after min on ice, the reaction tubes were centrifuged for min in a microfuge kept at °c. the resultant pellet was washed once with % ethanol, air dried, and resuspended in p of cdna synthesis mixture. primers for "pcr had the following sequences: the sequences of the oligonucleotide probes and "pcr primers were cdna synthesis the cdna synthesis mixture used to resuspend the extracted nucleic acids contained mm tris-hc , ph . , mm kc , mm mgcl,, mm dtt, . mm dntps, rnaguard ribonuclease inhibitor (pharmacia, u/reaction), m-mlv reverse transcriptase (gibco-brl, uireaction), and rtipcr-primer ( . pm). the reaction tubes were incubated a t °c for hr and then transferred to a n ice bath. a pl portion of each reverse transcription reaction was diluted to pl for "pcr amplification in reaction tubes containing mm tris-hc , ph . , mm kcl, . mm mgcl,, . mm dntps, taq polymerase (cambio, . ukeaction), bsa (brl, pg), and primers rtipcr-and pcr+ ( pm). a "touchdown" reaction cycle was used (don et al., ) : the steps were °c for sec, °c down to °c ( oc intervals) for sec and °c for min. there were two cycles for each annealing temperature down to °c followed by cycles a t °c. amounts ( pl) of the "pcr products were transferred to new tubes containing p of "pcr reaction mixture. except for the inclusion of only primers pcr+ and pv "pcr-(for pvnrt-pcr) or pcr+ and hrv ypcr-(for hrvnrt-pcr), the reagents were the ireland et al. same as those used in the "pcr. the reaction cycle was °c for sec, °c for sec, and °c for min. the cycle was repeated times. the reaction products were separated on . % agarose gels containing ethidium bromide and visualized by fluorescence in ultraviolet light. the "pcr products were then transferred to positively charged nylon membranes (amersham, hybond-n+ ) by alkaline blotting. filters were prehybridized for hr in x ssc, x denhardt's solution, % sds, and pg/ml of single-stranded herring sperm dna at the calculated melting temperature for the probe to be used. oligonucleotide probes were ' end-labelled using polynucleotide kinase (sigma) and [ y -~~p i a t p (amersham) as the precursor. labelled probes were separated from unincorporated atp on a column of sephadex g- (pharmacia). a labelled probe was added to the prehybridization mixture containing the filter and allowed to hybridize overnight a t the calculated t, of °c. after hybridization, the filters were washed twice in x ssc, . % sds for min at room temperature and twice in . x ssc, . % sds for min at the hybridization temperature. the filters were then autoradiographed on an x-ray film (kodak x-omat ar) overnight at - °c. rna was extracted from hrvs and enteroviruses (evs) of known serotype grown in cell culture. reverse transcriptase and "pcr primers (chosen from the conserved regions a and d shown in fig. ) were then used to give bp amplification products from these pvs [gama et al., , . after southern blotting, the "pcr products were probed with p-labelled oligonucleotide probes a t their calculated melting temperatures. the pv probe was a mer complementary to region c while the hrv probe was a mer complementary to region b [hyypia et al., . all the hrvs and coxsackie viruses gave the expected "pcr product as did six of the seven echoviruses. the absence of a reaction product from echovirus was expected [gama et al., ; hyypia et al., . the pv probe reacted with all the "pcr products except that from hrv . the hrv probe did not bind to the products from any of the evs but did bind to of the hrv "pcr products. the "pcr products from the evs and hrvs were later reamplified using the hrv probe as the reverse primer. none of the evs gave a product in this hrv nrt-pcr but all the hrvs, including those which had not given a positive result with the labelled probe, gave the expected ypcr product. the results for clinical samples are summarized in table i . nasal and throat swabs from leicester university volunteers with high symptom scores were chosen for analysis by pvnrt-pcr. no pvs had been cultured from these specimens although two hrvs and one echovirus had been isolated from other subjects in the study. seven positive swabs were detected in the "pcr and the products were analyzed by southern blotting and hybridisation to labelled probes. all seven reacted with the pv probe but only five with the hrv probe. when small amounts of the "pcr products were re-amplified using the pv probe as the reverse primer, five more positive swabs were detected. thus, out of ( %) nasal and throat swabs which were negative for pv by cell culture were positive when pvnrt-pcr was used. an adenovirus had been isolated from one of the remaining swabs and a further volunteer showed serological evidence of influenza b infection. thus the use of pvnrt-pcr to detect pvs increased the overall rate of pathogen identification from to %. pvnrt-pcr and hrv nrt-pcr were used to analyse nasal and throat swabs from participants in a cardiff university study ( from patients with colds and from healthy controls). the swabs were tested in random order and their identity was not known to the investigator until after the experiments were finished. swabs that gave positive results were extracted, reverse transcribed, and amplified again to confirm the findings. eleven of the patient swabs ( %) were positive for pv (six being amplified in both "pcrs and thus identified as hrvs) whereas none of the swabs from the healthy controls was positive by either nrt-pcr. only one of the patient swabs that was positive for pv when tested by pvnrt-pcr was also positive in cell culture. five of the pv-negative patient swabs had a respiratory pathogen identified by serology. two were positive for coronavirus, two for influenza a virus and one for adenovirus. the rate of pathogen identification was increased from to % by the use of pvnrt-pcr. pvnrt-pcr and hrv nrt-pcr were also to analyse nasal and throat swabs from subjects who had volunteered for a prospective study of respiratory infections in patients with asthma. sixty of ( %) swabs taken when the patients had colds were positive for pv when tested by pvnrt-pcr. all of the "pcr products were also reamplified using the hrv "pcr-primer and were thus identified as hrvs. thirteen of the nasal and throat swabs that were positive for hrv by hrv nrt-pcr were also positive for hrv in cell culture. there were three coinfections in which hrv was identified by hrv nrt-pcr and another virus was identified by serology. one subject had a coinfection with hcv oc and rs virus identified by serology. overall, the rate of pathogen identification was increased from to % by the use of pvnrt-pcr. convalescent nasal and throat swabs were taken a t least weeks after the acute swabs. three of convalescent swabs from subjects with hrv-positive acute swabs gave a further positive reaction. in evaluating the results of this study, consideration must first be given to the most commonly voiced criticism of pcr-its particular susceptibility to false positive results due to contamination [lo et al., . we attempted to identify any possible sources of contamination and adopt suitable procedures to eliminate them. a separate set of pipettors was used to prepare the mixtures of reagents and aerosol resistant pipette tips (continental laboratory products) were used throughout. different areas of the laboratory were used to add the " and ' "pcr substrates and to run agarose gels containing the pcr products. negative control cell cultures were included several times in every assay and never gave any product after extraction, reverse transcription and amplification. no carryover was observed, even when specimens containing large numbers of cell culture-grown virus particles were processed alongside these negative controls. when nasal and throat swabs from control subjects without cold symptoms were tested together with swabs from patients with colds, none of these control samples gave a positive result in the nrt-pcr assays. a simple rna extraction method is essential if many specimens are to be processed simultaneously for rt-pcr. gama and colleagues [ , used a method which required seven solvent extractions after the addition of carrier trna and vanadyl ribonucleoside complexes. we found that it was unnecessary to add ribonuclease inhibitors to our specimens or reactions if proteinase k in sds-containing buffer were added to the clinical material immediately after thawing. following enzyme digestion, we used three solvent extractions (one with phenolichloroform and two with chloroform) before ethanol/acetate precipitation of the nucleic acids. the pcrs for rhinovirus detection described by other groups of workers [gama et al., [gama et al., , hyypia et al., ; torgersen et al., had - cycles with annealing temperatures of " or °c. when these amplification conditions were used in this study, the dominant products were frequently various alternative fragments less than bp long. "touchdown" pcr was described by don and colleagues [ as a method of preventing such spurious priming during gene amplification. we used a "pcr in which the annealing temperature was decreased °c every second cycle from °c to a touchdown at °c at which temperature there followed more cycles. the production of spurious products was inhibited and it was then possible to use a nested primer in a "pcr to confirm the specificity of the reaction and to increase the sensitivity of the test. the oligonucleotide probes used in these experiments were not able to distinguish properly between evs and hrvs since the hrv probe had incomplete reactivity with the serotypes tested. this incomplete reactivity with hrv serotypes agrees with that observed by hyypia and colleagues using less stringent conditions in which the probe reacted with only out of clinical isolates of hrv. surprisingly, the hrv l b "pcr product did not hydridize with the hrv probe although a previously published sequence for nucleotides - of hrv l b shows perfect complementarity to this probe [torgersen et al., . variation may exist in the ' untranslated region for isolates of the same hrv serotype as it does for poliovirus serotypes [minor and dunn, . the problem of restricted reactivity with the hrv serotypes from cell cultures was overcome by using the same oligonucleotide as a "pcr primer with a n annealing temperature of °c. all the hrv "pcr products, including those that failed to hybridize with the probe, gave a "pcr product, whereas none of the evs was amplified. sommer and tautz [i found that primers with a length between and nucleotides needed a t least three homologous nucleotides at their '-end for successful priming but the remainder of the primer needed surprisingly little homology with the target sequence. although all of the pvs identified by pvnrt-pcr in the swabs from asthmatics could be further typed as hrvs by hrvnrt-pcr, five of the "pcr products from the cardiff subjects with colds were only reamplified with the pv and not with the hrv "pcr primer. mrc- and ohio hela cell cultures did not yield any virus from these swabs, which were all taken within a -day period. the patients may have been infected with one of several enteroviruses that have been associated with mild illness of the upper respiratory tract. these include coxsackie viruses a , , , , and b and ; and echoviruses , , , , and . it is also possible that the virus or viruses responsible were hrv(s) that were not recognized by the hrv "pcr-primer. this study has shown that pvnrt-pcr is a t least five times more sensitive than cell culture for the detection of pvs in nasal and throat swabs. pvnrt-pcr is also considerably faster since the whole process can be completed in less than two working days. amplification directly from clinical materials means that cell culture is unnecessary; nrt-pcr is then no more expensive than the traditional method of virus isolation. although all the cell culture-grown hrvs tested could be differentiated from other pvs using hrvnrt-pcr, it remains possible that this is not true for all hrvs. it is important that both pv and hrv primers are used in "pcrs for hrvs until a sufficiently large number of hrvs from clinical material have been identified by both this method and by cell culture. restriction enzyme analysis of hrv pcr products [torgersen et al., should enable us to determine whether or not the virus found in convalescent nasal and throat swabs is identical to that found in the corresponding acute specimens. in this way, we will gain information about the persistence of hrv infections. the fact that many specimens which were negative for pvs by cell culture were positive when tested by pvnrt-pcr has significant implications for future work on the epidemiology and potential therapy of respiratory tract infections. use of synthetic oligonucleotide probes to detect rhinovirus rna rhinoviruses. in fields bn touchdown' pcr to circumvent spurious priming during gene amplification evolutionary relationships within the human rhinovirus genus; comparison of serotypes , and polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction a collaborative report: rhinoviruses-extension of the numbering system from to the promises and pitfalls of pcr the nucleotide sequence of human rhinovirus b: molecular relationships within the rhinovirus genus polymerase chain reaction for human picornaviruses enzyme-linked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain false-positive results and the polymerase chain reaction the effect of sequences in the ' non-coding region on the replication of polioviruses in the human gut viruses as precipitants of asthmatic attacks in children rhinovirus and influenza type a infections as precipitants of asthma detection and differentiation of picornaviruses in clinical samples following genomic amplification primer-directed enzymatic amplification of dna with a thermostable dna polymerase minimal homology requirements for pcr primers minireview of chemotherapy of rhinovirus colds the complete nucleotide sequence of a common cold virus: human rhinovirus typing of human rhinoviruses based on sequence variations in the ' non-coding region prophylactic intranasal alpha interferon and viral exacerbations of chronic respiratory disease this work was in part supported by the cystic fibrosis trust, the bma hc roscoe fellowship, and the british lung foundation. we thank mrs. l. jacklin for expert technical assistance, dr. m. wiselka for help with the leicester university student study, and dr. a.p. smith, director, health psychology research unit, university of wales college of cardiff, for nasopharyngeal specimens from patients and controls. key: cord- - enjopig authors: li, yanpeng; deng, xilong; hu, fengyu; wang, jian; liu, ying; huang, huang; ma, jinmin; zhang, jianhui; zhang, fuchun; zhang, chiyu title: metagenomic analysis identified co-infection with human rhinovirus c and bocavirus in an adult suffering from severe pneumonia date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: enjopig • we first present a hrv-c and hbov co-infection in an adult woman with severe acute respiratory distress syndrome. • our study suggests that hbov co-infection might worsen the disease caused by hrv-c infection, and raises a question whether hrv-c-related severe pneumonia is often associated with co-infection of other respiratory viruses such as bocavirus. dear editor, human rhinovirus (hrv) and bocavirus (hbov) are two of the most common respiratory viruses associated with acute respiratory tract infections among children. , recent articles in this journal have highlighted the role of bocavirus and rhinovirus in potentially severe pneumonia in adults and younger children. , however co-infection with both viruses was very rare among adults. a previous study reported a -month child who was infected by hbov and also showed presence of hrv, developed severe pneumonia. here we reported co-infection with hbov and hrv-c in an adult woman with acute respiratory distress syndrome (ards). a -year-old woman caught a cold on jan. , , and developed symptoms of recurrent fever, cough, chills, expectoration, slight hemoptysis, muscle and joint pain in the following days (fig. a) . she felt chest tightness and shortness of breath on february , and visited guangzhou eighth people's hospital for further examination and treatment on february since her condition continuously worsened with cyanotic lips and pararthria. the patient had fever of . °c, heart rate of beats/ min, respiratory rate of breaths/min, blood pressure of / mmhg, and pulse oxygen saturation (spo ) of %. laboratory evaluation showed white blood cell count . - . × /l(normal, . - . × /l), lymphocyte . × /l (normal, . - . × /l), platelet count (plt) × /l (normal, - × /l), c-reactive protein . mg/l (normal, < . mg/l), hemoglobin (hb) g/l (normal, - g/l), oxygenation index (oi) mmhg (normal, - mmhg), cd t cell /mm (normal, - /mm ), cd t cell /mm (normal, - /mm ), and cd t cell /mm (normal, - /mm ) (supplementary table s ). auscultation of the lungs revealed extensive moist rales and computed tomographic (ct) scan showed bilateral diffuse infiltration (fig. a) . she was diagnosed with severe pneumonia with ards, and admitted immediately to the icu with high flow oxygen to maintain the spo at approximately % on february . during this period, she showed polypnea, dysphoria, frequent cough and slight hemoptysis. abundant hemorrhagic secretions were observed through bronchofiberscope. she was treated with antiviral and antibiotic therapies in combination with ulinastatin ( fig. a) . antiviral drug peramivir was used in the first three days, followed by oseltamivir in another three days. antibiotics (imipenem, cilastatin and moxifloxacin) were given for days. her body temperature returned normal on the fourth day and ct showed significant improvement in the patient's condition. she was switched to sequential non-invasive ventilation from february to , and then transferred to general ward and treated with low-flow oxygen and prednisone. the patient was discharged from the hospital on february and showed a sustained recovery in two subsequent visits on february and march . throat swabs and sera were collected during her hospitalization. blood and respiratory secretions were negative for bacteria, and fungi in cultures, and g-test and gm-test, respectively. hiv, hbv, influenza viruses, sars-cov, mers-cov and other coronaviruses were negative by elisa and/or (rt-)pcr assays. pulmonary secretions from the first day of hospitalization were further analyzed using metagenomic sequencing as described previously. of , , total reads, ( . %) were of viral origin. two respiratory viruses, hrv ( reads, . %) and hbov ( reads, . %) were found with high percentages (fig. b) . the presence of hrv and hbov were confirmed by specific (rt-)qpcr assays, and hbov rna was also detected at all sample points. hrv-c and hbov were determined by phylogenetic analyses of hrv vp /vp and hbov vp /vp fragments, respectively (fig. c) . specific igm and igg responses against hbov and hrv-c were further investigated by elisa (feiya biotech, china). the serum igg and igm levels of the patient showed . to . fold increases for hrv-c and . to . fold increases for hbov from february to february , respectively (fig. d) . above results indicated an acute co-infection with hbov and hrv-c in the patient. we further characterized the dynamics of both viruses using (rt-)qpcr assays (fig. e) . after days of treatment, the viral load of hrv-c and hbov among swabs showed a rapid decrease from . × copy/ml to below the detection limit, and from . × copy/ml to . × copy/ml, respectively. then, hrv-c viral load remained undetectable among swabs and sera, while hbov viral load started a slowdown from . × copy/ml to . × copy/ml during whole treatment, indicating a persistent infection of hbov . accompanied by the antiviral treatment, the patient progressively recovered and bilateral lung opacity improved rapidly, indicating that the treatment was effective. it was difficult to determine which virus was mainly responsible for the case. but the recovery of the patient from the rapid elimination of hrv-c and a persistent presence of hbov among swabs and sera suggested that hrv-c may be the principal causative agent for the severe pneumonia. hrv was often associated as severe respiratory illness in children and adults. [ ] [ ] [ ] as there are still questions about the role of hbov as a single causative agent, it is possible that the simultaneous hbov infections worsen the illness caused by preceding hrv-c infection. in this case, metagenomic sequencing showed a robust power to detect the causative agents of severe illness when the routine and traditional methods (e.g. culture, elisa, pcr, etc.) failed. after excluding bacterica, fungi, and major pneumonia-causing viruses (i.e. sars-cov, mers-cov, and h n ), we applied metagenomic sequencing to obtain the dna and rna sequences present in the sample, and determined the potential pathogens by similarity search with the sequence database covering all known viruses. the candidate viruses were further confirmed by specific (rt-)qpcr assays, phylogentic analysis, as well as seroconversion of igm and igg responses. using this pipeline, hrv-c and hbov were identified as the causative agents of this index patient with ards. in conclusion, our study reports a life-threatening pneumonia case caused by co-infection with hrv-c and hbov , and raises a question whether hrv-c-related severe pneumonia is often associated with co-infection of other respiratory viruses such as bocavirus. timeline of the patient's clinical course and evidence supporting the co-infection with hrv-c and hbov . a, timeline of the patient's clinical course and outcome. b, viral reads distribution. viral reads were classified by aligning again the ncbi viral sequence database with blast-n and blast-x. c, maximum-likelihood phylogenetic trees were generated by mega based on partial vp /vp sequences (~ bp) of bocavirus (left) and partial vp /vp sequences (~ bp) of rhinovirus (right). d, antibody responses to hbov and hrv-c at two time-points. results are shown as absorbance values at nm, cutoff . . difference between different groups was determined by the student's t-test. ** p < . . e, viral load dynamic of hbov and hrv-c in swabs and serums collected in different time-points. a high-resolution version of this slide for use with the virtual microscope is available as eslide: vm . no reported conflicts. human bocavirus: current knowledge and future challenges human rhinoviruses respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged months to years in a multi-country population sample genetic characterization of human bocavirus among children with severe acute respiratory infection in china primary and secondary human bocavirus infections in a family the eukaryotic gut virome in hematopoietic stem cell transplantation: new clues in enteric graftversus-host disease fatal respiratory infections associated with rhinovirus outbreak clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia guangzhou women and children's medical center supplementary data related to this article can be found online at https://doi.org/ . /j.jinf. . . . key: cord- -rs sd z authors: falsey, ann r.; branche, angela r. title: rhinoviruses date: - - journal: international encyclopedia of public health doi: . /b - - - - . - sha: doc_id: cord_uid: rs sd z human rhinoviruses (hrv) are ubiquitous pathogens and the leading cause of the common cold syndrome. hrv are very diverse with more than serotypes identified which cause disease in persons of all ages with the highest incidence documented in young children. although illness is typically mild and self-limited, lost time from work and school creates a considerable economic burden. infection of the upper airways is the most common site of infection, although lower airways disease is also well documented, as is the link between hrv infection and exacerbations of asthma. unfortunately, effective specific antiviral treatments and vaccines remain elusive. human rhinoviruses (hrv) are ubiquitous pathogens which are the leading cause of the common cold syndrome. once considered mostly a nuisance, these viruses are now appreciated as the cause of medically significant illness and a substantial burden of disease. although illness is typically mild and self-limited, lost time from work and school creates a considerable economic burden. infection of the upper airways is the most common site of infection, although lower airways disease is also now well documented, as is the link between hrv infection and exacerbations of asthma. unfortunately, effective specific antiviral treatments and vaccines remain elusive. in this article, the basic virology, pathogenesis of disease, epidemiology, clinical manifestation, and current methods of treatment and prevention will be reviewed. hrv is a member of the enterovirus genus within the picornaviridae family. since it was first identified in , serotypes of rhinovirus have been cultured and distinguished by distinctions in neutralizing antibody. these serotypes have been classified into two species, hrv-a and hrv-b, though in recent years sequencing technology has led to the identification of more than genotypes of viruses which do not grow in culture and have been placed into a third species, hrv-c (bochkov et al., ) . the rhinoviruses are also classified on the basis of receptor specificity. the 'major' receptor group, which comprises % of serotypes a and b, uses the intercellular adhesion molecule (icam- ) as the receptor for infecting host cells (staunton et al., ) . twelve hrv-a serotypes use an alternative site, the lowdensity lipoprotein receptor (ldlr), and the receptor for the hrv-c viruses is human cadherin-related family member (bochkov et al., ) . rhinoviruses are small, nonenveloped viruses with a single strand of positive-sense rna (greenberg, ) . each virion is composed of . kb of genomic material packed in a -nm icosahedric protein shell known as a capsid (royston and tapparel, ; figure (a)). the capsid is, in turn, comprised of pentamers each with five protein subunits called protomers containing four viral proteins, vp , vp , vp , and vp . the capsid is comprised of pentamers with a central shared depression or 'canyon.' (c) protomers are protein subunits of the pentamer containing four viral proteins, vp , vp , vp , and vp . vp proteins form a hydrophobic pocket in the canyon region which serves as the binding site for intercellular adhesion molecule (icam- ) receptors in host cells. each pentamer contains a central shared depression or 'canyon' in the five vp proteins of the protomer subunits, which serves as the binding site for icam- receptors in host cells (figure (b) and (c)). once attachment to the host cell is achieved, viral replication occurs in the host cell cytoplasm. antibody neutralization occurs when igg binds to the viral surface and obstructs access of the host-cell receptor to the viral attachment site at the base of the canyon (turner, ) . vp and vp are also found on the surface of the capsid and have antigenic sites important for the host immune response. vp is located on the internal side of the capsid and interacts with the viral genome. the genomic data that code for vp are somewhat conserved among the various subtypes of hrv, though variations in vp , vp , and vp account for antigenic diversity among serotypes and impede the development of effective vaccines and antiviral agents. for infection to occur hrv must reach the nasal mucosa of the susceptible individual. transmission from infected to susceptible individuals most likely occurs when viral load is highest and secretions are plentiful and difficult to control. under experimental conditions, a viral titer of tcid (tissue culture infective dose) was needed to transmit infection and most often occurred on the nd or rd day of a cold during peak symptoms (turner, ) . epidemiologic evidence suggests that direct contact and self-inoculation account for the majority of hrv transmissions. classic transmission studies of infected and susceptible subjects demonstrated that hand to hand or from hands to intermediary surfaces with recipient self-inoculation efficiently transmits hrv (hendley et al., ) . more recent experimental studies have shown that transmission of hrv is possible by small and large particle aerosols, but how frequently this occurs in natural settings is unclear. once the virus reaches the nasal mucosa, attachment to the epithelial cells occurs via the icam- receptor in % of infections or by the ldlr receptor depending on the serotype (pitkaranta and hayden, ) . hrv grows best at c in cell culture and primarily in the upper respiratory tract, although infection of the lower airways and sinuses, at higher body temperatures, is possible. both ciliated and nonciliated epithelial cells can be infected, and as infection progresses, affected areas may be patchy ( figure ). hrv infection is not cytopathic, and it is generally accepted that the majority of symptoms are due to the host inflammatory response. infection is limited to the respiratory tract with only rare reports of invasive disease (brownlee and turner, ) . although hrv is rarely cultured from blood, hrv rna has been detected in the plasma in % of normal children with hrv infection and was more common in those with severe illness and asthma exacerbations. however, it is unclear if detection of viral rna in plasma truly represents systemic infection. once infection has been established, a complex interplay of neurogenic and host responses occurs. early infection is characterized by a vigorous inflammatory response brought about by the elaboration of a variety of proinflammatory cytokines and chemokines, including interleukin (il)- b, il- , il- , and interferon-induced protein (pitkaranta and hayden, ; turner, ) . the concentration of these inflammatory mediators correlates with the severity of symptoms and results in an influx of neutrophils, increased vascular permeability, swelling of the nasal mucosa, and leakage of serum into nasal secretions. the kinins, bradykinin, and lysyl bradykinin can also be found in the secretions of hrv-infected individuals, and their presence and concentrations also correlate with symptoms. yet, treatment by blocking kinins has been ineffectual in alleviating symptoms raising questions as to the relevance of kinins in hrv disease pathogenesis. moreover, despite the frequency of antihistamines in many cold treatments, there is no evidence of histamine release in hrv infection. this may be due to the fact that neurogenic reflexes also appear to play a role in disease pathogenesis, with parasympathetic nerves controlling the flow of secretions from the nasal seromucous glands. finally, the importance of hrv infection and asthma exacerbations is now well recognized (gern and busse, ). the precise mechanism by which this occurs has yet to be fully investigated but appears to involve genetic predisposition and allergic airway factors measured by immunoglobulin e (ige). hrv-specific neutralizing antibody has been shown to be protective against infection and symptoms with homologous viruses (gwaltney et al., ) . infection results in the production of both serum igg and nasal iga beginning around weeks after infection and results in long-lasting protection to the specific infecting hrv serotype. resolution of symptoms and clearance of virus occur before the induction of nasal or serum antibodies, indicating a different mechanism for viral clearance. the inflammatory response produced during initial infection with hrv does not appear responsible for clearance of the virus. although the inflammatory response correlates with symptoms, asymptomatic individuals with little or no inflammatory response can efficiently clear hrv infection. the mechanism by which hrv clearance occurs likely involves the cellular immune response. both cd and cd cells can recognize heterologous hrv antigens and represent memory cells in persons with prior hrv infection (kennedy et al., ; steinke et al., ) . these cells function as immune surveillance and are able to produce a rapid adaptive immune response. hrv have worldwide distribution and circulate throughout the year. in temperate climates, peaks of viral activity are seen in the late summer and fall as well as the late spring (pitkaranta and hayden, ) . although hrv activity is relatively low during the summer months, it remains the most common cause of the common cold throughout the year. multiple serotypes can circulate simultaneously in the same geographic area, and no clear pattern of reappearance has been noted. households and schools are the most common places for spread of hrv infection. hrv affects persons of all ages with the highest incidence documented in young children. the incidence of hrv infection in children during the first years of life was noted to be . - infections per year in older studies using cell culture for viral detection (brownlee and turner, ) . more recently, winther et al. reported an average incidence of six picornavirus infections per year in a cohort of children aged months to years. colds remain common among working adults averaging two per year with hrv accounting for - % of illnesses and then appear to decline in frequency toward middle age (gwaltney et al., ) . although the incidence of acute respiratory tract infection declines with age, hrv infection can be a problem for older adults. in studies of community-dwelling older adults hrv was identified in % of illnesses reported by elderly persons followed for two winters in the united kingdom (nicholson et al., ) . additionally, hrv has been described as a cause of infection in older adults in day cares, long-term care facilities, and hospitalized with acute respiratory tract illnesses. asymptomatic hrv infection was recognized in the era of diagnosis by viral culture, but the widespread use of reverse transcription polymerase chain reaction (rt-pcr) for detection in recent studies demonstrates a high incidence of asymptomatic infection. approximately - % of asymptomatic subjects sampled are positive for hrv and approximately % of all hrv infections detected by rt-pcr are asymptomatic (brownlee and turner, ) . these findings make assessing the casualty of hrv with illness in epidemiologic studies challenging without the use of control subjects. the most frequent clinical syndrome ascribed to hrv is the common cold, an aggravating but usually self-limited illness. sir william osler characterized the illness particularly well in the principles and practice of medicine published in : "the patient feels indisposed, perhaps chilly, has a slight headache, and sneezes frequently. at first the mucous membranes of the nose is swollen, "stuffed up," and the patient has to breathe through the mouth. a thin, clear, irritating secretion flows, and makes the edges of the nostrils sore . usually within hours the nasal secretions become turbid and more profuse. and gradually, within - days, the symptoms resolve" (gwaltney et al., ) . in his studies, gwaltney et al. ( ) had detailed the symptoms and time course of the typical hrv upper respiratory infection (uri). rhinorrhea ( %) and sneezing ( %) were the most common complaints in the first days. scratchy or mild sore throat and headaches are also common in the first few days of illness. fever is uncommon, although some patients complained of feverishness ( %) and chilly sensations ( %). lower respiratory tract symptoms such as hoarseness and cough are less common occurring in one-fourth to one-third of cases although if present, symptoms tend to linger throughout the illness. when compared with other viral respiratory illnesses, significantly more sneezing occurred with hrv infections. the mean length of illness was . days with a range of - days. respiratory viral infection is felt to be a common predisposing factor for acute otitis media (aom). middle ear pressures have been shown to be abnormal in up to % of individuals with either experimentally induced or natural hrv infection (mcbride et al., ) . these abnormalities gradually abate over a -week period; however, swelling and obstruction of the eustachian tube may lead to the development of aom. a number of studies indicate that direct viral infection of the middle ear fluid is possible as hrv can be detected by viral culture ( - %) and pcr ( %) of middle ear fluids in infected individuals. thus, aom may be due to virus, secondary bacterial infection, or coinfection (greenberg, ) . primary viral rhinosinusitis appears to be a common phenomenon during uncomplicated natural infection with hrv. sinus abnormalities were observed on computed tomography (ct) scans in over % of adults with colds (gwaltney et al., ) . most commonly affected were the maxillary ( %) and ethmoid sinuses ( %), though the sphenoid ( %) and frontal sinuses ( %) could also be affected. hrv can often be detected by culture and rt-pcr in sinus brushings or aspirates from patients with viral rhinosinusitis, though the pathogenesis remains incompletely defined. the pressure created by nose blowing, sneezing, and coughing is felt to be an important factor in propelling hrv into the sinuses (greenberg, ) . importantly, in most patients, the abnormalities found on ct scans resolve within weeks without antibiotic treatment. because hrv replicates most efficiently at c, for many years infection of the lower respiratory tract at higher body temperatures was felt to be unlikely. recent studies have established hrv replication in the lower airways in natural and experimental infection. after experimental infection with hrv by inoculation of the upper respiratory tract, hrv was detected by in situ hybridization on bronchial biopsy during the peak of infection and - weeks later (papdopoulos et al., ) . consistent with this observation, hrv infection has been linked to bronchiolitis, exacerbations of asthma and chronic obstructive pulmonary disease (copd), and rarely pneumonia. hrv is an important trigger for asthma exacerbations in children and adults (gern and busse, ) . the peak of asthma exacerbations in the fall coincides with the increase in hrv activity as well as the start of the school year and seasonal allergen exposures. using rt-pcr in addition to standard testing, johnson et al. demonstrated that over % of the asthma exacerbations in children aged - years were associated with viral infections of which hrv accounted for approximately two-thirds. in addition to epidemiologic links of hrv and asthma, hrv human challenge models have provided insights into the mechanisms by which hrv infection may worsen asthma (greenberg, ; papdopoulos et al., ) . intranasal and inhaled inoculations of hrv induced bronchoconstriction after methacholine challenge in subjects with mild to moderate asthma (gern and busse, ) . the host immune response to infection induces proinflammatory mediators leading to a cascade of inflammation, activation of neural pathways to enhance airway hyperresponsiveness, and obstruction ( figure ). in addition to asthma, hrv has been associated with % of acute exacerbations of copd (greenberg, ) . in longitudinal studies of figure mechanisms of inflammation and asthma exacerbation triggered by human rhinovirus (hrv) infection. the host immune response to infection induces proinflammatory mediators leading to a cascade of inflammation that triggers airway hyperresponsiveness and obstruction. respiratory illness, hrv were the most commonly identified pathogens. for patients with moderate to severe copd, viral infections frequently lead to emergency room visits ( %) and hospitalizations ( %). the role of hrv as a cause of bronchitis or pneumonia in immunocompetent persons is unclear. a number of studies using either culture or rt-pcr have detected hrv in the upper airways in a small percentage of young children hospitalized with bronchiolitis or pneumonia (brownlee and turner, ) . hrv was detected in % of children < years of age hospitalized with bronchiolitis but was the sole pathogen in only %. similarly, % of children hospitalized with pneumonia had hrv found in secretions but over half had evidence of simultaneous bacterial infection. while there may be an association of hrv with these types of lower respiratory tract disease, the frequency of hrv in the general population makes assessing causality difficult. the evidence of lower respiratory tract infections with hrv in immunocompromised patients is more convincing (greenberg, ) . in a study of hospitalized immunocompromised patients, % developed fatal pneumonia. in six of seven total cases hrv was isolated from bronchiolar lavage fluid or endotracheal aspirates. persistent hrv in the lower respiratory tract of lung transplant patients has also been documented (brownlee and turner, ; ison et al., ) . although symptoms associated with 'the common cold' syndrome are often attributed to hrv disease, the clinical findings of rhinovirus infections are indistinguishable from those of other viral pathogens. a specific microbiologic diagnosis is most often made by detecting hrv in culture or with molecular assay and rarely using immunofluorescence or serotype-specific antibody assays. a variety of sample types from the upper airways have been used to confirm infection including nasal washes, nasopharyngeal swabs, and combined nose and throat swabs, though a few studies now convincingly report lower respiratory tract disease with virus detected in sputum and bronchoalveolar lavage samples from immunocompromised hosts (ison et al., ; papadopoulos et al., ) . for most patients, the timing of collection also plays a role in where virus is detected, with the highest concentration of virus typically detected in nasal fluids in the first - days when symptoms also peak (turner, ) . traditionally, viral culture has been used to identify hrv infection. viral isolation is accomplished by inoculation of virus on human embryonic fibroblast cells, incubation at c, and demonstration of cytopathic effect in the cell monolayers. in clinical practice, however, this proves to be a time-consuming method of diagnosis, requiring several days for virus isolation and yielding results usually after the acute phase of infection. consequently, microbiologic confirmation is rarely sought using culture methods. in contrast, the advent of new molecular techniques has led to a better understanding of the burden of disease associated with hrv infection with some studies demonstrating an increase in yield of three-to fivefold compared to standard virus culture (pitkaranta and hayden, ) . while not necessarily widely used in practice, rt-pcr has become the standard diagnostic tool and proven to be efficient, sensitive, and specific for detecting rhinoviruses. single-target hrv rt-pcr assays use primers that probe a conserved region of the hrv genome and are able to detect most serotypes (pitkaranta and hayden, ; turner, ) . however, hrv rt-pcr assays are currently only available commercially as part of multiplexed real-time pcr platforms for respiratory viruses such as the filmarray respiratory panel (biofire, utah). this has resulted in some loss of sensitivity and specificity since most of these assays are unable to distinguish between hrv and other enteroviruses. however, one potential benefit of using rt-pcr in practice is the ability to identify low-level viremia and asymptomatic shedding which may be important for infection control purposes in hospital wards with immunocompromised patients. rhinovirus antigen detection using elisa assays has been attempted but requires serotype-specific antibody and is therefore only of use in research settings. a single immunoassay has been developed which utilizes antibody against a conserved protein, the c protease of rhinovirus, but is unfortunately too insensitive for diagnostic use (turner, ) . similarly detection of serotype-specific neutralizing antibodies has been useful to diagnose natural hrv infections in family cohort and experimental virus challenge studies but is impractical for clinical use. treatment of hrv infections is supportive. the use of over-thecounter therapies may reduce symptoms of the common cold syndrome but are not specific to hrv infections. these remedies include antihistamines and nonsteroidal anti-inflammatory drugs which may alleviate symptoms without shortening duration of viral shedding and are also associated with sedation. other therapies reported to decrease symptoms, such as decongestants, saline nasal spray, expectorants, zinc sulfate lozenges, and high-dose vitamin c, have not been shown to be of significant benefit in randomized controlled trials (turner, ) . currently, there are no antiviral drugs approved for clinical use in hrv infections although a few agents have been advanced to clinical trials and shown modest results in decreasing either symptom severity or viral activity. leukocyte interferon was the first agent tested for activity against rhinovirus infection and demonstrated efficacy in preventing infection but not as treatment. however, the development of leukocyte interferon for prophylaxis was impeded by its prohibitively high cost and association with nasal toxicity (turner, ) . conversely, monoclonal antibody blockade of the icam- receptor, the site of cellular attachment for the majority of hrv-a and hrv-b serotypes, has also been studied and demonstrated a reduction in the severity of symptoms and viral shedding but failed to prevent infection in the rhinovirus challenge model (greenberg, ) . moreover, with the discovery of hrv-c viruses, which utilize a different cellular receptor, interest in this approach has waned. the hrv c protease cleaves the rhinovirus genome into individual structural and enzymatic components and is important to viral replication (turner, ) . it is highly conserved among rhinovirus serotypes and consequently a target for antiviral therapy. ruprintrivir is a c protease inhibitor which in experimental trial did not prevent rhinovirus infection but in clinical trial modestly reduced illness severity when treatment was initiated within day of infection (patick et al., ) . finally, the hydrophobic pocket formed by vp in the canyon region of the capsid, which facilitates attachment to the icam- receptors of host cells, has also been a target for drug development. capsid-binding agents bind to the hydrophobic pocket and prevent viral attachment to the cellular receptor and therefore viral replication. pleconaril is a capsid-binding agent with demonstrated antiviral activity against enteroviruses. in two large randomized, double-blind, placebo-controlled trials, patients with common cold symptoms were treated with pleconaril versus placebo within h of symptom onset greenberg, ) . compared to placebo, pleconaril treatment resulted in reduced illness duration by day and an overall decrease in the severity of symptoms. however, it was found to induce cytochrome p- a enzymes and therefore was not approved by the food and drug administration due to the potential for severe drug-drug interactions (greenberg, ) . new formulations of this agent are currently being considered for clinical trial. efforts to develop an effective vaccine for hrv infections have been unsuccessful to date, and strategies for preventing hrv infections are focused primarily on the interruption of transmission. since transmission occurs from direct contact, standard precautions including appropriate handwashing practices are likely the most effective preventive tool. in high-risk patients such as patients who have undergone recent stem cell transplantation, more stringent infection control practices including screening of symptomatic patients, early implementation of droplet and contact precautions, and restriction of visitors with upper respiratory tract symptoms have been shown to reduce rates of nosocomial infections (ison et al., ) . a number of handwashing agents have been assessed for their potential virucidal activity such as hand sanitizers containing % ethanol and treatments with acidic compounds turner, ) . none have demonstrated efficacy for the prevention of infection and are likely to be as effective as the use of soap and water. shortly after the discovery of rhinoviruses, serum serotypespecific neutralizing antibody and nasal hrv-binding immunoglobulin iga were shown to be protective against infection. efforts to develop hrv vaccinations soon followed, and initial clinical trials in humans involved the use of formalin-inactivated single serotype hrv vaccines. results of these trials demonstrated significant reduction in the rate of symptomatic colds (glanville and johnston, ) . however, with the discovery of hrv antigenic diversity resulting in more than different serotypes isolated, this approach was abandoned in favor of multivalent vaccines incorporating serotypes which unfortunately failed to demonstrate significant cross-protection among hrv serotypes. consequently hrv vaccine research was largely abandoned for more than years. recent advances in the development of a mouse modelfor hrv infection have permitted exploration of alternative approaches to immunization including induction of t cell-mediated immunity. in one recent report, recombinant vp protein (vp þ vp precursor), which appears to be highly conserved in hrv-a and hrv-b serotypes, was used as an immunogen in mice and shown to induce cross-serotype t cell recruitment and activation as well as neutralizing antibody production which resulted in accelerated clearance of virus in vivo (mclean, ) . this approach suggests that cross-serotype protection is possible, and while it is unlikely that a single immunogen will generate protection against all hrv serotypes, vp may be a useful candidate for a broadly protective subunit vaccine. hrv infect persons of all ages. infections in children can occur two to seven times a year and continue through life resulting in two to five symptomatic illnesses per year in adults. while most illnesses are mild and self-limited, in at-risk populations hrv infections may result in severe illnesses. consequently, though innocuously referred to as 'the common cold,' hrv infections constitute a significant burden with associated health care and economic costs. further study is needed to understand the pathogenesis of disease and adaptive immune responses associated with hrv infections which may also provide new targets for antiviral agents and broadly cross-protective vaccines. see also: influenza; pneumonia; respiratory infections, acute; respiratory syncytial virus; viral infections, an overview with a focus on prevention of transmission. cadherin-related family member , a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication new developments in the epidemiology and clinical spectrum of rhinovirus infections association of rhinovirus infections with asthma challenges in developing a cross-serotype rhinovirus vaccine respiratory consequences of rhinovirus infection rhinovirus infections in an industrial population. i. the occurrence of illness rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response computed tomographic study of the common cold efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of double-blind, randomized, placebo-controlled trials transmission of rhinovirus colds by self-inoculation rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia pathogenesis of rhinovirus infection alterations of the eustachian tube, middle ear, and nose in rhinovirus infection developing a vaccine for human rhinoviruses acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden rhinoviruses infect the lower airways in vitro antiviral activity of ag , a potent inhibitor of human rhinovirus c protease rhinoviruses: important respiratory pathogens rhinoviruses and respiratory enteroviruses: not as simple as abc a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses immune surveillance by rhinovirus-specific circulating cd þ and cd þ t lymphocytes rhinovirus a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection further reading how rhinovirus infections cause exacerbations of asthma key: cord- -knj rrut authors: albright, catherine j.; hall, david j. title: an exercise in molecular epidemiology: human rhinovirus prevalence and genetics date: - - journal: biochem mol biol educ doi: . /bmb. sha: doc_id: cord_uid: knj rrut human rhinovirus (hrv) is one of the most common human respiratory pathogens and is responsible for the majority of upper respiratory illnesses. recently, a phylogeny was constructed from all known american type culture collection (atcc) hrv sequences. from this study, three hrv classifications (hrva, hrvb, and hrvc) were determined and techniques for classifying new isolates of hrv were reported. the genetic change of this virus in specific populations over time is of great interest to understand the evolution and epidemiology of viruses. to facilitate the collections of hrv sequences over a number of years, a virology experiment was designed in which students test nasal lavage samples to look for hrv infection. students will learn a variety of techniques including rna isolation, cdna synthesis, qpcr, and agarose gel electrophoresis as well as bioinformatic skills though examination of sequences from the hrv‐field isolates. furthermore, students can look at symptom data from subjects to investigate correlations between symptom severity and factors such as stress and sleep patterns. such information can be used to examine hypotheses regarding hrv mutation, symptom severity and epidemiology. biochemistry and molecular biology education vol. , no. , pp. – , virology is an interdisciplinary field that incorporates aspects of biochemistry, cell biology, molecular biology, and immunology. thus, the study of virology is intriguing to students not only because of its depth but also because of viruses that have a very visible effect on a university population when cold and flu season arrives. viruses are parasitic nucleic acids, either dna or rna, and function through the ''hijacking'' of the host cell's own machinery. a productive viral infection must spread virus to other hosts to continue the viral life cycle, and the study of viral spread in a population is a principle concern of viral epidemiology. viral epidemiology not only examines how a virus functions in the host but also opens a window into human habits and interactions. some viruses are transmitted through aerosols, others via an oral/fecal route, and still others through the transmission of bodily fluids [ ] . thus, many viruses thrive by taking advantage of host habits. by understanding how viruses replicate and spread, students can gain insight into the functions of viruses, cells, and evolution. many viruses quickly adapt to environmental changes through error-prone replication, resulting in mutations and the generation of viral quasi species in which many genotypes coexist simultaneously during replication in the host cells. subsequent infection in a new host results in replication by viruses fit for a particular environment [ , ] . human rhinovirus (hrv), a picornavirus, is a member of the largest pathogenic virus family that includes such members as poliovirus, coxsackie virus, hepatitis a, and foot and mouth disease virus. picornaviruses are positive (mrna-like), single-stranded rna viruses, encased in a -nanometer icosahedral protein capsid. their genomes contain roughly , nucleotide bases that code for proteins [ , ] . hrv has a well-characterized genome with three main classifications currently identified: hrva, hrvb, and hrvc. during replication, millions of viral particles are made; however, the nucleic acids are not exactly identical. the hrv polymerase is error prone, and so several mutations occur through each round of replication. thus, each viral nucleic acid can be slightly different. these variants are referred to as viral quasi species and enable hrv to adapt to the host [ , ] . to date, there are hrv serotypes recognized by the american type tissue culture collection (atcc). although there are likely many more in the population considering the viral quasi species phenomenon. hrv infections occur worldwide at all times of the year, with strong outbreaks in the fall and late spring. however, hrv is only one of many respiratory viruses-others, including adenovirus and coronavirus, produce similar symptoms [ , ] ..hrv infections are usually limited to the upper respiratory tract, though they can also result in acute otitis (inflammation of the middle ear), sinusitis (inflammation of the paranasal sinuses), and lower respiratory tract infections [ ] [ ] [ ] . the extensive data available on hrv genomes enables many bioinformatics opportunities for students, including alignment of genome sequences to look for mutations at the rna level and differences among protein sequences. students can examine differences in the hrv serotypes over several years of data regarding a university population to identify hrv mutations that have occurred and their severity in causing symptoms in the host. this investigation is not only a way for students to study virus evolution but serves as an introduction in viral epidemiology. in this inquiry-based laboratory project, students use a variety of techniques, including rna isolation, cdna synthesis, qpcr, and agarose gel electrophoresis to look for the presence of hrv in nasal lavage samples from human subjects. the samples that are shown to be positive for hrv can then be sequenced to determine the strain of the isolate. by analyzing surveys in which subjects indicate severity of their symptoms, stress factors, and average hours of rest per night, students can identify possible contributors to hrv infection. these factors have been seen to make subjects more susceptible to the common cold and other viruses that cause upper respiratory infections [ ] [ ] [ ] [ ] . students who participate in this exercise should have basic biochemistry and molecular biology laboratory skills. they should be familiar with basic laboratory equipment (pipettors and centrifuges) and agarose gel electrophoresis if there is no qpcr thermocycler available to the lab. the laboratory exercise has convenient stopping points and can be completed in two laboratory periods if done efficiently. this laboratory works well with teams of two students who are allowed to divide the work. • students collect nasal lavage samples from themselves and classmates and survey their symptomsthis can be done before lab if student are equipped with needle-less syringes and sterile tubes and instructed in collection technique. • students isolate rna from samples and synthesize cdna (process can be stopped after isolation, but it is best to proceed to cdna step if possible). • students setup and perform both qpcr and pcrwhile these are running, students can analyze data from symptom surveys. • (students can run an agarose gel of the pcr product and image it.) • (students can run an agarose gel of the pcr product and image it.) • students prepare samples for sequencing. (session -when sequence data is returned) • students analyze sequences and draw conclusions regarding differences in serotypes found and how they differ from previous years. all aspects of the study involving human subjects must be approved by the school's institutional review board. subjects must be informed of the experiment and the use of the data and must sign an approved consent form before a sample can be obtained from them. students in the laboratory must be informed of the importance of anonymity of the samples they are testing, which should be assigned initials of the subject or, better yet, numbers. students should wear gloves whenever they are handling samples or cdna. special care should be taken when using trizol reagent-trizol should be used in a chemical fume hood to avoid inhalation of vapors. students should wear gloves and protective eyewear, avoiding any chemical contact with the skin or clothing. students first collected nasal lavage samples from themselves and their classmates. subjects were informed of the experiment and signed a consent form. students used a -ml syringe (becton dickinson, franklin lakes, nj) to inject ml of water into the nostril of the subject by placing the tip of the syringe just inside the nostril touching the inside of the nose and injecting the water. the water was then allowed to fall back out of the nose and collected in a sterile -ml centrifuge tube (cellstar-greiner bio-one, monroe nc) held under the nostril. students also asked their subjects to fill out a short survey (attached along with consent form) regarding their health, sleep habits, and stress levels. subjects rated symptoms associated with the common cold on a scale of to , with being nonexistent and being severe (survey included, see supporting information ). filtered pipet tips and gloves were used at all times to prevent rna degradation of the sample. from each sample, ll were moved to a sterile . -ml microfuge tube (eppendorf, hamburg, germany) with -ll nucleic acid extraction buffer (final concentrations of components: ph . tris mm, nacl mm, and edta mm) (supporting information ) and ll of carrier mix for rna isolation consisting of . ll of human dna ( ng/ll) (sigma-aldrich), ll glycogen ( lg/ll) (applied biosystems, carlsbad, ca), ll of glycoblue ( lg/ll; applied biosystems, carlsbad, ca) and . ll of phosphate buffered saline. rna was isolated from the sample by adding ll of trizol ls reagent (invitrogen, carlsbad, ca), and the tubes were vortexed (scientific industries inc, bohemia, ny) vigorously for minute before ll chloroform was added. the tubes were again vortexed for minute and then centrifuged (eppendorf centrifuge c, hamburg, germany) at , g for minutes. the aqueous layer was removed to a new eppendorf tube containing ll of rnasefree isopropyl alcohol. the tubes were then inverted several times, allowed to stand for at least minutes at room temperature and then centrifuged at , g for minutes to pellet the rna. the supernatant was then removed and ll of % ethanol, at room temperature, were added to the pellet. the tubes were then centrifuged for another minutes at , g, and the supernatant was carefully removed so as not to disturb the pellet of rna. each rna pellet was then resuspended in ll rnase-free water and vortexed vigorously to dissolve the rna. reverse transcription was conducted by first placing a mixture of the following reagents in each . -ml eppendorf tube: this first reaction was followed by a second pcr: the pcr contained ll of the platinum mix, ll of p - primer ( lm), ll of the p - ( lm), and ll of the products from the previous pcr. the products were analyzed in the following way: a . % agarose gel was used to separate the products of the pcr (seakem le, erie, pa; phusion, neb, ipswich, ma) hi-lo dna maker loading dye (promega, madison, wi). gels were made with tris borate edta (tbe) buffer and run at v (vwr power source v, west chester, pa). nucleic acids were imaged using ethidium bromide (etbr) and a fotodyne system (hartland, wi). hrv pcr products were sequenced to determine the genetic makeup of each field isolate. samples were prepared by adding ll of exo-sap-it (usbiochemicals, cleveland, ohio) into ll of pcr product. this mixture was incubated at c for minutes and then c for minutes. the resulting product was stored at c until sequencing could be performed. sequencing was performed by dewalch technologies, (houston, tx) using primer p - (acggacacccaaagtag; see supporting information ). sequences from the samples were entered into blast [ ] . the resulting blast information established to which hrv sequence from the atcc the sample was most closely related. a multiple sequence alignment and cladogram (using group tree) were both generated using web-based clustalw following the instructions provided [ ] . questionnaires for determining a subject's stress sleep and symptom severity were adapted from previously published studies [ , , , ] (supporting information ) . briefly, stress was assessed through a subject's rating his or her average stress level on a - scale with being the most severe. the quality and duration of sleep was assessed based on the reported number of hours ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the severity of symptoms was determined by subjects rating each of their cold symptoms on a scale of - ( most severe) and adding the resultant scores together to give a final symptom score. the symptom score was then adjusted to a - scale. a two-tailed t test was then used to determine the significance (graphpad software, inc http://www.graphpad.com/quickcalcs/ index.cfm). the primer set used to detect hrv in the samples was adapted from lee et al. [ ] and detects all possible atcc serotypes through the amplification of -bp portion of the noncoding region of hrv. the detection of hrv was conducted with both conventional pcr and qpcr using the same primer set. the qpcr method is preferable due to increased sensitivity. great care needs to be taken when preparing the viral rna from samples to prevent degradation and loss of the rna pellet during the precipitation procedure. of the samples obtained from human volunteers, the qpcr indicated were positive for hrv (fig. ) . the positive samples showed clear differences in the amount of viral nucleic acid present as indicated by the differences in cycle number. these differences can be manifest for several reasons including stage of the infection and the efficiency of hrv nucleic acid isolation. one can quantify the exact amount of virus using laboratorygrown strains, but this approach is beyond the scope of this article and the availability of equipment in many undergraduate laboratories. the standard pcr and subsequent agarose gels demonstrated this technique's weaker sensitivity compared to the qpcr (figs. a and b) . of the positives on qpcr, six were positive with standard pcr. samples with low amounts of viral nucleic acid or samples contaminated with rnases likely only appear as positive on the qpcr. no samples appeared as positive in conventional pcr after a negative result from qpcr. there are hrv serotypes recognized by the atcc and extensive data available on hrv genomes enables many bioinformatics opportunities for students, including alignment of genome sequences to look for changes at the rna level. standard pcr was used to generate enough genetic material for sequencing, and students retrieved sequences from the service provider website and submitted them as blast queries to identify to which atcc strain their hrv samples were most similar [ ] . the analysis revealed that two of the six samples were variants of hrv with % of the sequence identical with the atcc reference strain. the other samples were identified as being similar to atcc reference strains hrv , hrv , and hrv . the sequences were aligned with each other and atcc reference strains using clus-talw and a group tree cladogram was generated (fig. ) [ ] . from a research perspective, the sequence data is most interesting because, as samples are sequenced yearly, it will be possible to create a genetic database of hrv strains found in a college-aged population and to do further analysis on many levels. indeed, a discussion was initiated with the students on how the nucleotide variations between their samples and the atcc strains may influence the ability of the virus to elude the immune system, replicate or spread between hosts. additional avenues of investigation can include the analysis of predicted rna secondary and tertiary structure [ , ] and the sequencing of the nucleic acid responsible for coding of the viral capsid or the entire genome. with this information and a free program called vigor, students can identify all viral proteins and perform bioinformatics analysis in comparison to atcc reference strains [ ] . in a sense, this laboratory is similar to the program sponsored by howard hughes medical institute called ''phage-hunters'' [ ] . fig. . quantitative pcr of nasal lavage samples. qpcr spectra indicating hrv presence in samples. twenty-eight total samples were run- of subjects who reported being sick with common cold symptoms and who reported no symptoms. the x-axis indicates the number of qpcr cycles, and y-axis indicates the relative fluorescence units. dashed lines are those subject who were hrv positive but did not report being sick. the qpcr was as described in methods and was performed by lawrence university students during an undergraduate general biology class. fig. . standard pcr of nasal lavage samples. pcr was conducted as described and products were subjected to gel electrophoresis and stained with sybr green. (a) agarose gel indicating no successful hrv standard pcr in lanes , , , . successful standard pcr amplification for sequencing of qpcr samples in lanes , , , and . lanes and correspond to hrv and hrv positive controls, respectively. (b) agarose gel indicating no successful hrv standard pcr in lanes , , , , , , and . successful standard pcr amplification for sequencing of qpcr samples in lanes and . lane corresponds to the hrv positive control. standard pcr and subsequent agarose electrophoresis was done as described in methods and was performed by lawrence university students during an undergraduate general biology class. studies have shown that the severity of experimental viral infections depends not only on the viral strain but also on the sleep and stress levels of the individuals involved [ ] [ ] [ ] [ ] . in this study, of the students volunteering samples chose to participate in the symptoms, stress, and sleep surveys as well (figs. a- c ). only of the hrv positive subjects chose to participate in the surveys while all of the subjects that tested negative for hrv participated in the surveys. of those subjects negative for hrv, five were found to have reported being sick. in this instance, other viruses are probably responsible for the observed symptoms. this laboratory can easily be extended to include pcr primers to identify other viruses including influenza, corona virus, and adenovirus among other possibilities [ ] . the average symptom, sleep, and stress scores were compared between hrv positive and hrv negative subjects and between reported sick and healthy subjects (figs. a- c) . in our data, there are two significant trends as determined by an unpaired t test. first, symptom scores correlated positively with being hrv positive (p ¼ . , not significant) or sick (p ¼ . , significant; fig. a ). second, the sleep amount correlated positively with being hrv negative (p ¼ . , not significant) or healthy (p ¼ . , significant; fig. b ). however, there was no apparent relationship between self-reported stress level and having hrv or being sick (fig. c) . a larger n value can be achieved if this laboratory is taught over multiple years, and data is analyzed in aggregate. in addition to looking for correlations between symptoms, stress levels, amount of sleep and infection, students can collect information regarding subject residences and activity participation. students can group subjects by dormitory and when the results of the sequencing return, see whether or not different residences on campus harbor different hrv strains. however, great care must be taken to ensure student privacy in the data collection and encoding process. the value in doing this laboratory is not only pedagogical; in teaching it yearly and sequencing to track the infectious cycle of hrv, it will be possible over the years to build a record of what serotypes of hrv are affecting a population, with particular symptoms, at a given location. this information can help in answering questions such as: what types of mutation occur? what types of hrv are prevalent? what percentage of the respiratory infections is hrv? who contracts hrv? how does this correlate to sleep habits and stress levels? by creating such a dataset, insight can be gained regarding hrv infection and mutation in a college population. principles of virology the authors thank sally berben, ronald f. peck, michael schreiber, and wai-ming lee for technical assistance and critical reading of the manuscript. rna degradation is the single most troublesome area. although positive and negative samples can easily be identified via qpcr, getting enough pcr products for the detection by gel electrophoresis can be hampered by rna degradation. rnase-free reagents should be used at all times, and gloves are a must. furthermore, delays in the procedure for isolating the rna can result in degradation. often, running a third pcr can be helpful in these situations. this extra step can be done by using ll of the platinum mix, ll of p - primer ( lm), ll of the p - ( lm), and ll of the products from the previous pcr. key: cord- -if f ii authors: yuan, lijuan; geyer, annelize; saif, linda j title: short-term immunoglobulin a b-cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with wa human rotavirus date: - - journal: immunology doi: . /j. - . . .x sha: doc_id: cord_uid: if f ii immunological memory is important for protecting the host from reinfection. to investigate the development and sites of residence of intestinal memory b cells, and their role in protective immunity to reinfection with an enteric virus, we assessed the association between memory b cell and antibody-secreting cell (asc) responses and protection using a gnotobiotic pig model for human rotavirus (hrv) infection and diarrhoea. the isotypes, quantities and tissue distribution of rotavirus-specific memory b cells and asc were evaluated prechallenge ( and postinoculation days [pid]) and postchallenge ( postchallenge days [pcd]), using enzyme-linked immunospot (elispot) assay, in gnotobiotic pigs inoculated once with virulent or three times with attenuated hrv and challenged at pid with the corresponding virulent hrv. complete protection against hrv shedding and diarrhoea was associated with significantly higher numbers of immunoglobulin a (iga) and immunoglobulin g (igg) memory b cells and asc in the ileum of virulent hrv-inoculated pigs at challenge. in contrast, pigs inoculated with attenuated hrv had lower numbers of iga and igg memory b cells and asc in intestinal lymphoid tissues, but higher numbers in the spleen. the bone marrow had the lowest mean numbers of iga and igg memory b cells and asc prechallenge in both groups of hrv-inoculated pigs. therefore, bone marrow was not a site for iga and igg rotavirus-specific antibody production or for memory b cells after inoculation with live rotavirus, from pid up to at least pid. the effect of in vitro antigen dose was examined and it was determined to play an important role in the development of asc from memory b cells for the different tissues examined. rotavirus is a major cause of dehydrating diarrhoea in humans and animals. elucidation of the immunological mechanisms by which the host is protected against infection and disease is critical for the development of successful vaccines. the recent possible association between the use of a live oral rotavirus vaccine in infants and cases of intussusception suggests that more detailed information about immune responses to live oral rotavirus vaccines is needed to improve the safety of such vaccines. in previous studies, , we reported that the number of rotavirus-speci®c immunoglobulin a (iga) antibody-secreting cells (asc) present in the intestinal lamina propria of gnotobiotic pigs at the time of challenge (primary asc) correlates with protection against infection and diarrhoea when challenged with human rotavirus (hrv). however, the number of immunoglobulin g (igg) asc in systemic lymphoid tissues does not correlate with protection. , the majority of speci®c iga asc at mucosal surfaces induced by primary exposure to intestinal virus infection are short-lived, with a life span, in general, of a few days. ± therefore, the number of long-lived plasma cells and virus-speci®c memory b cells induced by primary antigen exposure play important roles in protection against reinfection. , long-lived plasma cells are terminally differentiated cells and do not proliferate upon exposure to recall antigen. the cells persist without the requirement for antigen presence and secrete antibodies continually in the secondary lymphoid organs where the cells reside. in contrast, memory b cells do not secrete antibodies spontaneously. when activated by recall antigen, memory b cells differentiate rapidly into asc and secrete greater quantities of antibodies with higher af®nity compared to naõ Ève b cells. , , memory b cells differ from naive b cells in several ways (which may also be related to their role in conferring protection) including: ' lower requirements for activation (from antigen, cytokine and t-cell help); ' their capacity to present antigen directly to t cells; and ' their capability to colonize antigen-draining sites, including the mucosal epithelium. a detailed understanding of the relationship between the development of asc and memory b-cell responses, the major resident sites of memory b cells after rotavirus infection, and the association of asc and memory b-cell responses with protective immunity against reinfection, is needed for the development of ef®cacious vaccines. williams et al. reported that at ± weeks after oral inoculation of adult mice twice with the ec strain of murine rotavirus, the majority of rotavirus-speci®c iga and igg asc and memory b cells were localized in the intestinal lymphoid tissues, with much fewer numbers of iga asc and memory b cells in bone marrow. the bone marrow was suggested to be one of the major sites for long-term antibody production. in mice, after a single rotavirus infection, high titres of rotavirus-speci®c antibodies persist and the mice are protected from reinfection for life. this life-long immunity is not observed after rotavirus infections in humans, who continue to be susceptible to reinfection with rotavirus, but such reinfections usually occur as subclinical infections after subsequent re-exposure. in humans, reinfections with the same serotype of rotavirus also occur. lack of long-term immunity to rotavirus raises the question as to the role of memory b cells in protective immunity to rotavirus infections in humans. to help address this question, we studied memory b-cell responses to hrv in a gnotobiotic pig model of hrv infection and disease. the gnotobiotic pig is a unique model for the study of immunity to hrv induced by natural infection and by rotavirus vaccines, because the pathogenesis and immunity of rotavirus infections in pigs and humans are similar, but differ from the subclinical infections and life-long immunity produced in adult mice. ± in addition, the gastrointestinal physiology and the development of a mucosal immune response by gnotobiotic pigs closely resemble those of human infants. the objectives of this study were as follows: ( ) to optimize the detection of memory b cells by enzymelinked immunospot (elispot) assay. ( ) to evaluate the isotypes, quantities and tissue distribution of rotavirus-speci®c memory b cells and to compare them with virus-speci®c asc responses in gnotobiotic pigs inoculated with virulent wa hrv (mimic natural infection) or attenuated wa hrv (mimic oral rotavirus vaccines). ( ) to assess the association between asc and memory b-cell responses and protection against rotavirus infection and disease. near-term pigs were derived by hysterectomy and maintained in sterile isolation units, as described previously. to study the effects of the dose of in vitro stimulating antigen on the induction of asc from memory b cells, eight gnotobiotic pigs ( ± days of age) were orally inoculated with < ¯u orescent focus-forming units (ffu) of the virulent wa strain of hrv (serotype p a[ ]g ). the % infectious dose (id ) of the virulent wa hrv inoculum for gnotobiotic pigs is j ffu. of the eight gnotobiotic pigs used, four were killed on postinoculation day (pid) and four were orally challenged with < ffu of virulent wa hrv on pid and killed on postchallenge day (pcd) . to study asc and memory b-cell responses to wa hrv and their association with protection, gnotobiotic pigs were assigned to one of three groups. group (eight pigs) was inoculated (at ± days of age) orally once with < ffu of the virulent wa hrv. group (nine pigs) was inoculated three times (on pid [ ± days of age], pid and pid ) with < ffu of the tissue culture-adapted attenuated wa hrv strain. giving three doses of attenuated wa hrv was to mimic the immunization regimen of a live reassortant rotavirus vaccine tested in human infants. the inoculation dose of attenuated wa hrv was determined in a previous study, based on the ®nding that at least two doses of < ffu virus were needed to induce seroconversion in % of pigs. group (®ve pigs) was mock inoculated with diluent (minimal essential media; gibco life technologies, grand island, ny) three times in the same time frame as group . a subset of pigs from each group was challenged with < ffu of virulent wa hrv at pid . rectal swabs were collected daily for days after inoculation and for days after challenge. the presence of virus in rectal swab¯uids was analysed by antigen-capture enzyme-linked immunosorbent assays (elisas) and by cell culture immuno¯uorescent infectivity (ccif) assays to determine virus shedding and infectivity, respectively, as previously described. , the pigs were observed daily for diarrhoea. faecal consistency was scored as follows: , normal; , pasty; , semiliquid; and , liquid. pigs with daily faecal consistency scores of i were considered diarrhaeic. to assess longer-term immune memory after rotavirus infection, four pigs were inoculated with one dose of < ffu of virulent wa hrv at days of age and kept in isolation units for weeks. serum samples from the pigs were collected at selected time-points over the entire period. isotype-speci®c antibody titres to wa hrv in the serum were determined by an elisa, and virus-neutralizing (vn) antibody titres were measured by using a plaque-reduction assay, as described previously. these four pigs were killed on pid to evaluate the levels of longer-term asc and memory b-cell responses to wa hrv. small intestinal contents (sic) and large intestinal contents (lic) were collected from these pigs and the isotype-speci®c antibody titres to wa hrv in sic and lic were determined by elisa. isolation of mononuclear cells the intestinal and systemic lymphoid tissues, including duodenum, ileum, mesenteric lymph nodes (mln), spleen, peripheral blood lymphocytes (pbl), and bone marrow were collected from all pigs when killed on pid , pid (pcd ) or pid . mononuclear cells (mnc) from the lymphoid tissues were isolated as previously described. , bone marrow was collected from the femurs of pigs by¯ushing the bone marrow cavity with ml of ca + -and mg + -free hanks' balanced salt solution (gibco brl). single-cell suspensions were prepared by passing the hanks' balanced salt solution± bone marrow suspension through stainless steel -mesh screens of a cell collector (cellecter; e-c apparatus corp., st. petersburg, fl). the mnc were isolated from bone marrow by ficoll±hypaque (ficoll-paque . ; sigma chemical co., st. louis, mo) density-gradient centrifugation, similar to the isolation procedure used for mnc from peripheral blood. the puri®ed mnc from all tissues were resuspended in complete medium consisting of rpmi- (gibco brl) supplemented with % fetal bovine serum, mm hepes (n- -hydroxyethyl-piperazine-nk- -ethanesulphonic acid), mm l-glutamine, mm sodium pyruvate, . mm non-essential amino acids, mg/ml of gentamicin, mg/ml of ampicillin and mm -mercaptoethanol (e-rpmi). rotavirus from infected ma cell-culture supernatants (titre < ffu/ml) was semipuri®ed by centrifugation ( rg) through a % (wt/wt) sucrose cushion. the viral pellets were suspended to < : of the original volume of cell-culture supernatant in . m tris buffer (ph . ) containing . m nacl and . m cacl (tnc), aliquoted, and stored at x u. the protein concentration of the preparations was determined by spectrophotometric analysis (gene-quant; pharmacia, lkb, biochrom, cambridge, uk). mock antigen from uninfected ma cells was prepared and stored in an identical manner. in vitro stimulation of mnc puri®ed mnc were restimulated in vitro with semipuri®ed attenuated wa hrv antigen, by using methods previously described , for determining the number of memory b cells in lymphoid tissues of rotavirus-sensitized pigs. the duration of antigen stimulation and the dose of the semipuri®ed virus antigen added to the mnc cultures were optimized based on ®ndings that initially established the day and dose that yielded the greatest number of asc in the elispot assay. memory b-cell responses were compared to a range of antigen doses ( . ± mg and ± mg/ . r mnc in two experiments). six micrograms of semipuri®ed wa hrv antigen was used for the study of memory b-cell responses to virulent and attenuated wa hrv. the semipuri®ed wa hrv antigen or mock antigen was added to duplicate wells of each mnc preparation ( . r mnc in . ml of e-rpmi per well of a -well tissue culture plate [corning glass works, corning, ny]) and incubated at u in an atmosphere of % co . at the second, third and fourth day of incubation, . ml of fresh e-rpmi medium was added to each well. on the ®fth day, mnc from two to four duplicate wells were pooled, rinsed once with rpmi- and diluted to . ml with e-rpmi. the viable mnc numbers were counted by trypan blue exclusion. the mnc were used in elispot assays for enumeration of virus-speci®c asc that were derived from memory b cells during incubation in the presence of the recall antigen. elispot for virus-speci®c asc an isotype-speci®c elispot assay for enumerating immunoglobulin m (igm), iga and igg rotavirus-speci®c asc , was used to evaluate effector and memory b-cell responses to wa hrv. the elispot assay performed on the day of mnc extraction was used to determine the numbers of in vivo antigen-activated asc (e.g. plasma cells), because plasma cells secrete antibody spontaneously. the elispot assays performed after the mnc were stimulated with rotavirus or mock antigen in cell culture for days were used to determine the numbers of memory b cells. memory b cells were identi®ed on the basis of their ability to proliferate, differentiate and secrete antibody upon stimulation with the recall wa hrv antigen. brie¯y, the concentrations of viable mnc recovered from cell culture were adjusted to r /ml ( . r mnc in . ml of erpmi). when fewer than . r viable mnc were harvested, the existing number of mnc were used in the assays. wa hrv-infected, ®xed-cell plates were washed ®ve times with distilled water prior to use. single-cell suspensions of mnc from each tissue were added to duplicate wells with r , r and r mnc/well for the elispot assay. the plates were incubated for < hr at u in a % co atmosphere and then washed and incubated with biotinylated mouse monoclonal antibody (mab) (puri®ed ascites¯uids) to pig igg (derived from hybridoma h , . mg/ml), pig iga (derived from hybridoma d , . mg/ml), or pig igm (derived from hybridoma c , . mg/ml), for hr at room temperature. plates were washed and horseradish peroxidaseconjugated streptavidin (kirkegaard & perry laboratories, inc., gaithersburg, md) was added (diluted : ). after incubation for hr at room temperature, the plates were washed and blue spots were developed with tetramethylbenzidine and the h o peroxidase substrate system (kirkegaard & perry laboratories, inc.). the numbers of virus-speci®c asc were determined by counting blue spots in the wells and reported as the number of virus-speci®c asc per r mnc. because of the low viability of mnc from some of the tissues, the number of memory b cells from the tissues with fewer than . r viable mnc were adjusted according to the viable mnc numbers at the time the in vitro elispot assays were performed to compensate, and to enable comparison, of the memory b-cell responses among tissues. in this case, the numbers of memory b cells were calculated as: ascr( r }numbers of viable mnc). fisher's exact test (sas institute inc., cary, nc) was used to compare proportions of pigs with diarrhoea and virus shedding among groups. a one-way analysis of variance (anova) was used to compare mean duration and days of onset of virus shedding and mean duration of diarrhoea. the asc numbers were compared among or within groups using the kruskal± wallis rank sum (non-parametric) test. statistical signi®cance was assessed at p< . for all comparisons. inoculation of pigs with one dose of virulent wa hrv conferred complete protection; inoculation with three doses of attenuated wa hrv conferred partial protection against hrv-associated diarrhoea ( % protection rate) and virus shedding ( % protection rate), upon challenge (table ) . among nine pigs inoculated with attenuated wa hrv, only one shed virus on the th day after the ®rst inoculation, indicating the limited intestinal replication of the attenuated wa hrv in pigs. after challenge at pid ( ± days of age), all mock-inoculated control pigs shed virus and developed diarrhoea, although the severity was slightly reduced compared to the neonatal nai È ve pigs infected with virulent wa hrv at ± days of age (table ) . this is possibly because of the slightly reduced susceptibility of older pigs to severe rotavirus diarrhoea. the viable mnc were counted at the initiation of the mnc cultures and after days of incubation with the hrv stimulating antigen or mock antigen. the viability of cells on the initial day of mnc incubation were > % in all of the tissues. after days of culture in the presence of mg of stimulating antigen, variations in viabilities were seen within and between the tissues, between the time-points and among the groups (data not shown); there were no statistically signi®cant differences among the mnc viabilities. however, notable differences for virulent, attenuated wa hrv, or mockinoculated pigs at any given time-point included the following: ( ) peripheral blood and mln had the highest mean viabilities (< %); ( ) duodenum and ileum had lower mean viabilities (< %), which may re¯ect the requirement for a higher stimulating antigen dose for these tissues; and ( ) mnc from mock-inoculated pigs had the lowest viabilities (< %) in the ileum. to optimize the in vitro cell culture conditions and the detection of memory b cells, the effect of different antigen doses on the viability of mnc was evaluated, with ileum, mln and spleen from pigs inoculated with virulent wa hrv in initial experiments. the doses of in vitro-stimulating hrv antigen of . ± mg did not alter substantially the viability of mnc from ileum, mln and spleen. the mnc cultured with mock antigen had similar viabilities to the mnc cultured with the lowest concentration of antigen tested ( . mg per . r mnc) (data not shown). however, increased antigen doses of ± mg were associated with a twofold increase of viability (from % to %) of mnc from ileum at day of incubation (data not shown). the speci®city of the in vitro elispot assay for quantifying memory b cells was examined and con®rmed by the results of the following experiments: ( ) no rotavirus-speci®c asc were detected from the naive animals (two mock-inoculated pigs killed on pid ) even though their mnc were stimulated with mg of rotavirus antigen in vitro for days. ( ) the numbers of asc detected by the in vitro elispot assay increased with increasing doses of hrv stimulating ) ( . *determined by enzyme-linked immunosorbent assay (elisa) and cell culture immuno¯uorescence infectivity assays. the proportion (percentage) of asc detected from mnc cultured with mock antigen divided by the mnc stimulated with hrv antigen were all< %, except for . % for iga asc in ileum at one time-point in the dose±response experiment (data not shown). these observations indicate that > % of asc detected by the in vitro elispot assay were not longlived plasma cells. instead, they were asc derived from the proliferation and differentiation of memory b cells induced by inoculation of the pigs with rotavirus. for the attenuated wa hrv-inoculated pigs, the asc detected by the in vitro elispot assay may have included a small percentage of plasma cells, as a subpopulation of b cells that were primed in vivo by the repeated inoculations and destined to become plasma cells could have developed into asc spontaneously during the in vitro cell culture. the few long-lived plasma cells (< % in ileum and < % in other tissues) detected in the mock antigen-stimulated wells, or the few plasma cells from attenuated wa hrv-inoculated pigs, were not subtracted from the numbers of asc detected from viral antigen-stimulated wells because of the statistical insigni®cance of their small percentage compared with the major asc populations derived from memory b cells in each tissue. the asc numbers detected in the elispot assay on the day of mnc extraction (without in vitro culture and antigen stimulation) were reported as asc; the asc numbers detected in the elispot assay after in vitro antigen stimulation were reported as memory b cells (and may also include a small percentage of plasma cells) in the present study. the effects of antigen dose in vitro on the detection of memory b cells were evaluated using a wide range of wa hrv antigen ( . ± mg per . r mnc). the numbers of igm, iga and igg asc in all of the tissues tested (ileum, mln and spleen) increased according to increasing doses of in vitro stimulating antigen within the range of . ± mg of rotavirus antigen per . r mnc (fig. ) . the magnitude of the increases in the number of iga and igg asc were highest for cells from the ileum ( . -to . -fold) and lowest for cells from the spleen ( . -to . -fold). when the antigen doses were increased from to mg/ . r mnc, the effect of the stimulating antigen dose in each tissue varied. the numbers of iga and igg asc increased slightly according to increasing doses of antigen for cells from the ileum. however, they decreased for cells from the mln and remained similar for cells from the spleen (fig. ) . the asc responses to wa hrv are depicted in fig. , fig. and table . before challenge (pid [pcd ]), the mean numbers of virus-speci®c iga and igg asc, respectively, in the duodenum ( -and -fold), ileum ( -and . -fold) and mln (®ve-and -fold), were signi®cantly higher in virulent wa hrv-inoculated pigs than in the attenuated wa hrvinoculated pigs, and were associated with a % protection rate in this group upon challenge (fig. ) . after challenge (pid [pcd ]), there were no increases in the numbers of virus-speci®c igm, iga or igg asc in this group of pigs, indicating little or no in vivo viral antigen restimulation (fig. ) . in contrast, in the attenuated wa hrv-inoculated pigs, anamnestic iga and igg asc responses were detected in most of the tissues, indicating reinfection postchallenge. the numbers of iga asc increased in the duodenum (signi®cantly), ileum, mln and spleen (from ®ve-to -fold). the numbers of igg asc increased signi®cantly in the duodenum, ileum, mln and spleen (from -to -fold) (fig. ) . the hrv-speci®c iga asc predominated prechallenge in the duodenum of pigs inoculated with either virulent (iga/ igg ratio= ) or attenuated (iga/igg ratio= ) wa hrv. in contrast, igg asc predominated prechallenge in the ileum of virulent (iga/igg ratio= . ) or attenuated (iga/ igg ratio= . ) wa hrv-inoculated pigs. after challenge, iga asc still predominated in the duodenum (iga/igg ratio= . ) of virulent wa hrv-inoculated pigs. conversely, in the attenuated wa hrv-inoculated pigs, igg asc predominated (iga/igg=ratio . ) in the duodenum and iga/igg ratios increased to . in the ileum at pid (pcd ). the igg asc predominated in systemic lymphoid tissues of pigs inoculated with virulent or attenuated wa hrv pre-and postchallenge, except in the spleen of virulent wa hrv-inoculated pigs postchallenge where the iga/igg ratio was < (fig. , fig. ) . no virus-speci®c asc were detected in mock-inoculated pigs until after challenge when igm asc predominated in all tissues at pcd (fig. ) . memory b-cell responses to wa hrv, detected in vitro by the elispot assay, are depicted in fig. , fig. and table . in the virulent wa hrv-inoculated pigs, memory b-cell responses were high in the ileum but low in the duodenum, both pre-and postchallenge (fig. , fig. ). the numbers of iga and igg memory b cells were signi®cantly higher ( -to -fold) in the ileum of virulent wa hrv-inoculated pigs compared to attenuated wa hrv-inoculated pigs on pid (pcd ) and pid (pcd ) (fig. and table ). in virulent wa hrvinoculated pigs, the highest mean numbers of iga and igg memory b cells were detected in the ileum; however, in attenuated hrv inoculated pigs the highest mean numbers of iga and igg memory b cells were detected in the spleen before challenge. the igg memory b cells predominated in all of the tissues of pigs inoculated with virulent or attenuated wa hrv, both pre-and postchallenge. in virulent wa hrv-inoculated pigs after challenge, the mean numbers of iga and igg memory b cells increased, although not signi®cantly, in the duodenum, ileum (iga only), mln, spleen and peripheral blood. in the attenuated wa hrv-inoculated pigs, the mean numbers of iga and igg memory b cells did not change substantially and were lower than in the virulent wa hrv-inoculated pigs in all tissues postchallenge (fig. , fig. ). few memory b cells were detected in the bone marrow of either virulent or attenuated wa hrv-inoculated pigs before and after challenge (fig. , fig. and table ). therefore, the bone marrow was not a site of resident memory b cells of any isotype on pid (pcd ) or pid (pcd ). the numbers of rotavirus-speci®c asc and memory b cells in the longer-term virulent hrv-inoculated pigs decreased substantially on pid (data not shown) compared to pid in the pigs inoculated with virulent hrv. the mean numbers of iga and igg asc in all tissues were fewer than eight per r (fig. ) . the vn antibody titres in serum peaked at pid (gmt ) (fig. ) . at pid , the gmt of iga antibody in the serum was , comparable to the antibody titres in the sic (gmt ) and lic (gmt ) at this time; however, the igg antibody titres in the serum were -fold higher than those in the sic (gmt versus ). igg antibodies were not detected in the lic on pid . knowledge regarding the mechanisms involved in the generation, anatomical localization and persistence of memory b cells, particularly in regard to mucosal surfaces, is scarce. to improve our understanding of memory b-cell responses to enteric pathogens, the present study focused on the role of antigen (virulent versus attenuated wa hrv) in determining the isotype and localization of memory b cells, the tissue origin of memory b cells and the association of memory b-cell responses with protective immunity to reinfection against an enteric virus. inoculation of pigs with virulent or attenuated wa hrv induced different isotype, magnitude and localization patterns for asc and memory b-cell responses. virulent wa hrv induced signi®cantly higher numbers of iga and igg asc in the duodenum, ileum and mln compared to attenuated wa hrv, before challenge. rotavirus-speci®c iga and igg memory b cells were detected in the duodenum, ileum, mln and spleen, but with much higher numbers in the ileum and slightly higher numbers in mln than in the other tissues on pid and . therefore, memory b cells induced by virulent wa hrv resided primarily in the ileum. for the pigs table . ( . ( ) ( ) ( . ( ) ( . ) inoculated with attenuated wa hrv, although the highest mean number of iga asc was in the duodenum, the highest mean numbers of igg effector and iga and igg memory b cells were in the spleen. the lower numbers of iga and igg asc detected in all tissues of this group of pigs on pid (pcd ) re¯ect the lower antigenicity of the attenuated wa hrv; virulent wa hrv is known to replicate more extensively than attenuated wa hrv throughout the small intestine. however, high numbers of igg memory b cells detected in the spleen after inoculation with attenuated wa hrv indicate that naive b cells in systemic lymphoid tissues were primed by the viral antigen. the original site of priming of the memory b cells detected in the systemic lymphoid tissues was not determined in this study. they could be nai È ve b cells activated in situ in the systemic lymphoid tissues, or b cells primed in the intestines that then migrated into the spleen and resided there. the systemic igg memory b-cell responses and the low numbers of intestinal iga and igg asc were associated with only partial protection of these pigs when challenged on pid . this observation agrees with our previous ®ndings for pigs inoculated intramuscularly with inactivated wa hrv or inoculated intranasally with / -virus-like particles, in which high numbers of igg asc and memory b cells were induced in the systemic lymphoid tissues. however, minimal or no protection was conferred by inoculation with the inactivated wa hrv or / virus-like particles. these ®ndings re-emphasize our theory that protection against an enteric virus infection is dependent not only on the magnitude, but also on the site and isotype, of the asc and memory b-cell responses. , , predominant iga asc responses were detected in the duodenum of virulent wa hrv-inoculated pigs; however, the majority of the virus-speci®c iga memory b cells were resident in the ileum, not the duodenum. in pigs, as in humans, the duodenal lymphoid tissue consists mainly of effector sites (lamina propria). , the ileal lymphoid tissue consists of both effector sites (lamina propria) and the major inductive sites (peyer's patches). , the asc responses detected in the duodenum and ileum were presumably attributed to lymphoid cells in the lamina propria because no plasma cell development occurs in peyer's patches. , the anatomical difference between the duodenum and ileum of pigs may explain the different patterns of asc and memory b-cell responses detected. for mice, it has been suggested that the memory b cells committed to iga production and which originate in inductive sites (peyer's patches and mln) , are also resident in these sites weeks after intramuscular immunization. other studies of mice showed that rotavirus-speci®c iga memory b cells were detected in both peyer's patches and the small intestinal lamina propria, but with a delayed appearance in the lamina propria. , the distribution and magnitude of the memory b-cell isotypes between peyer's patches and the ileal lamina propria were not examined in the current study because of the dif®culty in isolating and separating mnc from the peyer's patches and ileal lamina propria of gnotobiotic pigs. therefore, the ileal mnc originated from both types of lymphoid tissues in the ileum. in contrast to several studies perfomed in mice, , ± the results from this study demonstrated that bone marrow is not a major site of residence for primary effector and memory b cells in pigs on pid , and after oral inoculation with hrvs. our ®nding concurs with a recent study of mice by c.a. moser & p.a. of®t (personal communication) showing that no rotavirus-speci®c iga or igg asc or memory b cells were detected in bone marrow after adult mice were inoculated orally with rhesus rotavirus (rrv) from pid to pid . although williams et al. reported, in their study of adult mice orally inoculated twice with murine rotavirus ec strain, that rotavirus-speci®c asc were detected at pid ± in the bone marrow, the absolute numbers of rotavirus-speci®c asc in the lamina propria were more than -fold higher than those in bone marrow. it appears that the few asc detected in the bone marrow may have been asc in the peripheral blood traf®cking through the bone marrow (the asc numbers in the peripheral blood were not reported in this study). similarly, williams et al. reported that rotavirus-speci®c,¯uorescence-activated cell sorter (facs)-separated a b iga memory b cells, adoptively transferred from donor mice seven months after oral ec rotavirus inoculation into rag- mice, preferentially resided in the lamina propria with asc numbers at least -fold higher than in bone marrow. in pigs, the bone marrow showed a similar or lower magnitude of asc responses compared to that seen in the peripheral blood. other research teams , ± have reported that bone marrow was the major site of long-term antibody production after oral inoculation of mice with cholera toxin, after an acute systemic infection of mice with lymphocytic choriomeningitis virus (lcmv), , and in mice systemically immunized with the t-cell-dependent antigen, ovalbumin. because of limitations in the length of time that gnotobiotic pigs can be maintained in isolation units, longer-term b-cell responses beyond pid were not evaluated in this animal model. however, very few asc or memory b cells were detected in bone marrow, on pid , or , of pigs inoculated with virulent wa hrv. thus, our data and the studies of mice infected with rrv (ref. ; c.a., moser & p.a. of®t, personal communication) suggest that bone marrow is not an inductive or a resident site for primary effector and memory b cells after a heterologous rotavirus infection of pigs (hrv) or mice (rrv). differences between replicating organisms and non-replicating antigens, and the target tissues and replication sites for a systemic virus (lcmv) versus an enteric virus (wa hrv), could be the main reasons for the divergent responses observed. attenuated wa hrv that replicated to only a low extent in the intestinal epithelia, stimulated mainly igg memory b cells that were resident in spleen. interestingly, however, the igg memory b cells induced by virulent wa hrv were resident in both the ileum and spleen, with the latter tissue and not the bone marrow the predominant site on pid . the iga memory b cells detected in the spleen on pid of pigs inoculated with virulent hrv may explain the successful use of the parenteral route of inoculation to boost mucosal antibody responses and protection, as observed in studies of orally primed pigs and humans. , in the longer-term study of pigs inoculated with virulent wa hrv, the serum iga antibody titres started to decline after pid ; however, the serum igg antibody titres were maintained at peak levels from pid to pid . our previous study has also shown that high titres of virus-speci®c igg antibodies, but not iga antibodies, persist in the serum for prolonged time-periods after inoculation with either virulent or attenuated wa hrv. these ®ndings suggest that the spleen is a major site for long-term serum igg antibody production in rotavirus-inoculated pigs. signi®cant waning of iga memory b-cell responses from pid to pid in the intestine of pigs inoculated with virulent wa hrv, indicates a short b-cell memory to rotavirus infection in pigs. these results agree with previous observations that iga memory to mucosal pathogens was short lived and needed periodic boosting to be maintained, as often occurs after repeated re-exposure to endemic enteric pathogens in the environment. , in contrast, memory b-cell responses to murine rotavirus edim in mice at weeks postinoculation were even higher than those at weeks postinoculation. it will be of interest to assess the association between b-cell memory responses and protective immunity against reinfection in pigs at pid ; however, we could not challenge gnotobiotic pigs with hrv at this timepoint owing to the limitation in the size of the isolation units and the potential reduced susceptibility of older pigs to rotavirus diarrhoea for assessment of postchallenge protection beyond pid . optimization of the elispot assay for the detection of memory b cells was carried out using dose±response studies. the in vitro antigen dose played an important role in the development of asc from memory b cells. the viability of mnc in the present study was very similar to that of a previous study of conventional pigs infected with enteric or respiratory coronaviruses, in which the viabilities of mnc from the spleen and mln were ± %, and for the duodenum and ileum were ± %, on day of culture. the wide variation in viability of mnc among tissues in the present study prompted us to quantify the amount of in vitro antigen that would adequately promote memory b-cell proliferation and differentiation for each tissue. results from dose±response studies showed that an antigen dose of mg was optimal in our elispot assay system for the detection of memory b cells in the mln and spleen, but slightly less optimal for the detection of memory b cells in the ileum. when the wa hrv antigen dose was increased from mg to mg, iga and igg memory b-cell numbers in both spleen and mln started to decline, indicating that memory b cells in these two tissues were suf®ciently stimulated and that the lower numbers of iga memory b cells detected in the mln of pigs inoculated with attenuated wa hrv compared to those in the spleen and peripheral blood was not because of underestimation by poor in vitro antigen stimulation. the possible selective expression of systemic homing receptors (e.g. a b , l-selectin) on b cells activated by attenuated wa hrv could explain the greater asc responses in the systemic lymphoid tissues versus intestine in this group of pigs. a fourfold higher dose of restimulating antigen resulted in a twofold increased viability and similarly increased the numbers of iga and igg memory b cells from the ileum of pigs inoculated with virulent wa hrv. this ®nding suggests that the memory b cells in the ileum had a lower af®nity to the recall antigen or a higher threshold for reactivation than the memory b cells in the spleen and mln. if the higher antigen dose were used, slightly higher memory b-cell numbers might have been detected in the intestinal lymphoid tissues; however, it would not alter the conclusions drawn from the present results, as the ileum showed the strongest memory b-cell responses among all the tissues. the requirements for different antigen doses for the induction of optimal iga and igg memory b-cell responses from different lymphoid tissues may be explained by the assumption that the immune regulatory cells (including cd + , ab and cd t helper cells, and antigen-presenting cells) are distributed and function differently in different sites in the immune system. in conclusion, inoculation of gnotobiotic pigs with virulent or attenuated wa hrv induced different localization patterns of asc and memory b cells. substantial iga b-cell memory was established in the ileum, and later in the spleen, but not in the duodenum or bone marrow of gnotobiotic pigs inoculated with virulent wa hrv. the magnitude of the iga and igg asc responses in the intestinal lymphoid tissues and iga memory b-cell responses in the ileum, but not igg asc or memory b-cell responses in the systemic lymphoid tissues, was associated with protective immunity in pigs inoculated and challenged with virulent wa hrv. our ®ndings on the sites of resident memory b cells generated in response to an enteric viral infection parallel recent ®ndings on the distribution of memory t cells. , , lika a substantial subset of t-cell memory, short-term b-cell memory also resides in nonorganized lymphoid tissues such as the ileal lamina propria. fields virology. philadelphia: lippincott-raven publishers advisory of committee on immunization practices (acip) systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus production of lymphocytes and plasma cells in the rat following immunization with human serum albumin lifetime of plasma cells in the bone marrow autoradiographic studies on the immune response. . the kinetics of plasma cell proliferation on immunological memory memory b cells are biased towards terminal differentiation: a strategy that may prevent repertoire freezing contributions of memory b cells to secondary immune response memory, but not nai È ve, peripheral blood b lymphocytes differentiate into ig-secreting cells after cd ligation and costimulation with il- and the differentiation factors il- , il- , and il- memory b cells from human tonsils colonize mucosal epithelium and directly present antigen to t cells by rapid up-regulation of b - and b - the memory b cell subset responsible for the secretory iga response and protective humoral immunity to rotavirus expresses the intestinal homing receptor the gnotobiotic pig as a model for studies of disease pathogenesis and immunity to rotavirus development of a mucosal rotavirus vaccine intranasal administration of / -rotavirus-like particles with mutant escherichia coli heat-labile toxin (lt-r g) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs procurement and maintenance of germfree swine for microbiological investigation pathogenesis of an attenuated and a virulent strain of group a human rotavirus in neonatal gnotobiotic pigs development of mucosal and systemic lymphoproliferative responses and protective immunity to human group a rotavirus in a gnotobiotic pig model porcine pararotavirus: detection differentiation from rotavirus, and pathogenesis in gnotobiotic pigs ef®cacy of an orally administered modi®ed-live porcine origin rotavirus vaccine against postweaning diarrhea in pigs serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease enumeration of isotype-speci®c antibody-secreting cells derived from gnotobiotic piglets inoculated with porcine rotaviruses contribution of immune responses induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge limiting dilution analysis of virus-speci®c memory b cells by an elispot assay production and characterization of monoclonal antibodies to porcine immunoglobulin gamma, alpha, and light chains relationship between expression of iga by peyer's patch cells and functional iga memory cells immunology of the porcine gastrointestinal tract the response of gut-associated lymphoid tissue in gnotobiotic piglets to the presence of bacterial antigen in the alimentary tract peyer's patches: an enriched source of precursors for iga-producing immunocytes in the rabbit induction of mucosal b-cell memory by intramuscular inoculation of mice with rotavirus relative importance of rotavirus-speci®c effector and memory b cells in protection against challenge persistence of intestinal antibody response to heterologous rotavirus infection in a murine model beyond year long-term antibodies after an oral immunization with cholera toxin are synthesized in the bone marrow and may play a role in the regulation of memory b-cell maintenance at systemic and mucosal sites bone marrow is a major site of long-term antibody production after acute viral infection long-lived plasma cells: a mechanism for maintaining persistent antibody production passive immunity to coronavirus and rotavirus infections in swine and cattle: enhancement by maternal vaccination different secretory immunoglobulin a antibody responses to cholera vaccination in swedish and pakistani women induction and persistence of local rotavirus antibodies in relation to serum antibodies group a rotavirus veterinary vaccines mucosal immunity memory t cells ± local heroes in the struggle for immunity lefranc Ëois l. preferential localization of effector memory cells in nonlymphoid tissue visualizing the generation of memory cd t cells in the whole body we thank zhiqian fan, viviana parren Ä o, marli s. p. azevedo, cristiana iosef, peggy lewis and paul nielsen for technical assistance. we also thank john vancott (children's hospital medical center, cincinnati, oh, usa) for helpful comments. this work was supported by grants (ro ai and ro ai ) from the national institutes of health. salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. key: cord- - mnsm s authors: ahanchian, hamid; jones, carmen m; chen, yueh-sheng; sly, peter d title: respiratory viral infections in children with asthma: do they matter and can we prevent them? date: - - journal: bmc pediatr doi: . / - - - sha: doc_id: cord_uid: mnsm s background: asthma is a major public health problem with a huge social and economic burden affecting million people worldwide. viral respiratory infections are the major cause of acute asthma exacerbations and may contribute to asthma inception in high risk young children with susceptible genetic background. acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma. discussion: while the importance of preventing viral infection is well established, preventive strategies have not been well explored. good personal hygiene, hand-washing and avoidance of cigarette smoke are likely to reduce respiratory viral infections. eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, such as om- , may reduce recurrent infections in susceptible children. there are no practical anti-viral therapies currently available that are suitable for widespread use. summary: hand hygiene is the best measure to prevent the common cold. a healthy balanced diet, active probiotic supplements and immunostimulant om- may reduce recurrent infections in asthmatic children. asthma is a major public health problem with a huge social and economic burden affecting million people worldwide [ ] . viral respiratory infections are the major cause of acute asthma exacerbations and contribute to asthma inception in high risk young children with susceptible genetic background. a history of wheeze associated with respiratory viral infections early in life is one of the major risk factors for the later development of asthma [ ] [ ] [ ] [ ] [ ] [ ] , together with sensitization to aeroallergens in early life and a family history of asthma and allergies, reflecting a genetic predisposition. respiratory viral infections are also the principal cause of asthma exacerbations in children and adults [ ] [ ] [ ] [ ] [ ] [ ] . however, the question of whether viral infections "select" susceptible hosts or whether viral infections may induce asthma de novo by "damaging" airways is not settled. in other words, do viruses cause or simply unmask asthma? respiratory viruses first infect nasal epithelial cells which triggers an antiviral response. this response is driven by type i (α/β) and iii (λ) interferons (ifn) that are induced following recognition of viral ribonucleic acid (rna) by pattern recognition receptors (prrs). toll-like receptors (tlrs) are cell surface and endosomal prrs, whilst the rna helicase receptors (rig-i and mda- ) and nodlike receptors (nod ), detect viral rna in the cytoplasm. signalling via the prrs activates transcription factors (irf- , irf- , nf-κb), which lead to the production and secretion of type i and iii ifn. the ifns then bind to cell surface receptors to activate a separate pathway leading to the production of interferon stimulated genes (isgs) which encode antiviral proteins that combat infection, as well as prrs and transcriptional factors which further amplify ifn production. the respiratory syncytial virus (rsv), human meta-pneumovirus (hmpv) and human rhinovirus (hrv) are all single stranded rna viruses but engage differently with cell signalling pathways. in airway epithelial cells rsv and hmpv rna are primarily detected by rig-i in the cytoplasm [ , ] . rsv can also be detected by nod [ ] . hrv is endocytosed by epithelial cells, and is therefore primarily detected by tlr in the endosome early in the infection process and by rig-i and mda- later in infection following upregulation of these prrs [ ] . the fusion (f) protein of rsv is recognised by tlr at the epithelial cell surface [ ] . a successful antiviral response would see the infection limited to the upper airway, as is the case clinically with the majority of viral infections in healthy individuals. should such a response be deficient, then predominantly upper-airway viral infections, such as hrv, may spread to the lower airways, causing lower respiratory symptoms and an exacerbation of asthma in predisposed individuals. while definitive data are yet to be produced, experimental hrv infections in adult volunteers initially suggested that asthmatics were more likely to develop lower respiratory infections (lri) than healthy adults, i. e. less likely to be able to limit viral replication to the upper airways [ , ] . subsequent in vitro infection of primary airway epithelial cells from asthmatic and healthy adults with hrv have demonstrated that asthmatic cells produce less ifn-β [ ] and ifn-λ [ ] making them potentially more susceptible to infection, slower to clear infection, and more susceptible to virus-induced cell cytotoxicity. deficiencies in the ifnα response of peripheral blood mononuclear cells and plasmacytoid dendritic cells from asthmatic adults and children has also been observed, in these particular studies, in response to rsv, hrv [ , ] and influenza a [ ] . it is likely that the overall impaired innate immune response of the asthmatic airway epithelium is a result of deficiencies in the antiviral response of both epithelial cells and immune cells. childhood, especially infancy, is characterized by developmentally-regulated deficiencies in innate and adaptive immunity [ ] . such deficiencies are likely to increase the risk of viral lri in children, especially in those at high risk for asthma and allergies. each year, at the end of summer, parents of asthmatic children are concerned about acute asthma exacerbations following a common cold, asking how to minimize the risk during the winter viral season. it is a valid concern as up to % of asthmatic children have an intermittent or wheeze which is mostly symptomatic after viral infections [ , ] . asthmatics with exacerbationprone phenotype are susceptible to acute exacerbations requiring hospitalization or an unscheduled visit for medical attention. major risk factors for acute exacerbations include previous acute exacerbation, allergy, young age, poorly controlled asthma, and, in particular, viral respiratory infections. moreover, recent data suggests an interaction between allergies and viral infections occurs to increase the risk of asthma exacerbation [ ] . acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma [ , ] . viral respiratory infections are the main cause of asthma exacerbations in children ( - %) and are a major risk factor for admission in hospital every autumn [ ] [ ] [ ] . hrv are the most common viral agents [ ] ; other respiratory tract viruses detected in children with an asthma exacerbation include rsv, influenza, coronavirus, hmpv, parainfluenza virus, adenovirus, and bocavirus [ ] [ ] [ ] . current drugs for the prevention and treatment of virus-induced exacerbation of asthma are poorly effective and novel alternative therapies are needed. much research interest has focused on the potential role respiratory viral infections play in the inception of asthma. it is well established that hospitalization for rsv bronchiolitis is a risk factor for asthma during childhood [ , ] . epidemiological studies have shown an increased risk of asthma with lri caused by hrv. in the childhood origin of asthma (coast) birth cohort study, wheezing with rsv (odds ratio [or], . ), hrv (or, . ), or both hrv and rsv (or, ) was associated with increased asthma risk at age six years [ ] . the childhood asthma study (cas) in perth, australia showed that wheezing with hrv or rsv in the first year of life was a risk factor (or, . ) for current wheeze at five years of age [ ] . infant birth about four months before the winter virus peak carried the highest risk of developing asthma compared with birth months before the peak [ ] . the risk of asthma is increased by severe lri (slri), especially in the presence of allergic sensitization in early life [ , ] . there appears to be a synergistic interaction between viral infection and allergic sensitization, suggesting a "two hit" model for induction of persistent asthma. these data also provide a series of novel strategies for the primary prevention of asthma by prevention of either allergic sensitization or of slri in high risk children. this strategy is also supported in a study by simoes et al. [ ] , in which the use of palivizumab to prevent rsv infection decreased the risk of recurrent wheezing in nonatopic premature infants. the crucial period, with respect to asthma initiation, appears to be the first two to three years of life during which the growth and remodelling of lung and airways proceeds at maximum rates. pulmonary inflammation resulting from atopy and slri occurring during this vulnerable time is hypothesized to perturb underlying tissue differentiation programs, resulting in deleterious long term effects on respiratory functions. as a result, there is widespread belief amongst the paediatric respiratory community that intervention measures that can lower the frequency and/or intensity of slri in early life amongst the high risk atopic subgroup of children are likely to be successful at preventing asthma. if successful, these strategies would have major implications for reducing the high impact of this chronic disease on the community [ , , ] . recent studies using culture-independent techniques have challenged the long-held dogma that lungs are sterile and have demonstrated that a microbiota community exists in the lung [ ] [ ] [ ] . the implications of these new data are not clear, however new concepts and more research is required. the resident microbiome is different in the presence of respiratory disease [ , ] ; therefore interactions between respiratory viruses and the resident pulmonary microbiome are postulated. the pulmonary and gastrointestinal microbiota influence the immune system and interventional approaches (by bacterial immunostimulants, prebiotics and/or probiotics) to create a healthy gut and respiratory microbiota are potential strategies for the prevention of viral infections [ ] . children are important vectors for hrv transmission to family members particularly siblings [ , ] . hrv shedding peaks two to four days after infection and decreases sharply thereafter, although nasal samples can be positive for rhinovirus for up to five weeks after a symptomatic infection [ ] . there are three ways of common cold transmission in children. first, inhalation of small particles aerosolized by coughing; second, large particle droplets from saliva expelled while sneezing; and third, self-inoculation of one's own conjunctivae or nasal mucosa after touching a person or object contaminated with the cold viruses. the first two methods are inefficient [ ] , while the third is the most important method of transmission. the mode of transmission could differ with age of the index case, duration of contact, and other factors. moreover, there is some evidence that the daily activities of infected people can lead to the contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets [ ] . meticulous hand hygiene is the best measure to prevent the common cold; frequent hand washing and avoid touching one's nose and eyes [ ] [ ] [ ] . the use of alcohol-based hand sanitizers is also effective [ , ] . the promotion of handwashing was associated with a - % reduction in respiratory-tract infections and colds in child-care centres in the usa [ ] canada [ ] and australia [ ] and a % decrease in absences due to respiratory illness in the school setting [ ] . hand hygiene campaigns were also successful in reducing absenteeism caused by influenza-like illnesses among schoolchildren in egypt [ ] . similar programs within families would be expected to reduce transmission of hrv between family members. a recent cochrane review which included data from randomised controlled trials and observational studies, investigated the effectiveness of physical interventions to reduce the spread of respiratory viruses. the authors concluded that respiratory virus spread can be reduced by hygiene measures (such as handwashing), especially around younger children and can reduce transmission from children to other family members [ ] . controversy still exists and a newly published study showed that an antiviral hand treatment used by adult volunteers, recruited from a university community, did not significantly reduce rv infection or rv-related common cold illnesses [ ] . asthmatic children should avoid close contact with people who have colds especially during the first three days of their illness. there is little evidence to support the effectiveness of face masks to reduce the risk of viral respiratory infections and consequently, the use of mask is generally not recommended for prevention of common cold [ , ] . immune function and anti-viral defenses have a number of components, both specific and non-specific. asthmatic children can improve their immune function by following some simple advice including a healthy life style with regular exercise, a balanced diet, adequate sleep and avoiding environmental tobacco smoke, stress and unnecessary antibiotics. exercise has anti-inflammatory effects and in the long term can protect the development of chronic diseases and obesity [ ] . regular exercise of moderate-intensity is associated with a reduced incidence of upper respiratory tract infection. however, long hours of intensive training appear to make children more susceptible to upper respiratory tract infections [ ] [ ] [ ] . the recommended means of aerobic exercise is walking, with an optimal frequency of three to five days a week and an optimal duration of to minutes of continuous activity [ ] . in a recent study, the iga secretion rate was negatively correlated with the incidence of infections [ ] . a recent randomized trial comparing meditation and exercise with wait-list control among adults aged years and older found significant reductions in ari illness [ ] . malnutrition is the most common cause of immune deficiency worldwide and a balanced diet is fundamental for a healthy immune system. vitamin d deficiency has been associated with increased risk of infections, earlylife wheeze and reduced asthma control [ , ] . vitamin a derivatives are involved in the regulation of the immune system and tissue inflammation as well as prevention of respiratory infections [ ] . zinc, selenium and other trace elements are necessary for function of both innate and adaptive immune function. a high intake of fruit and vegetables ensures adequate consumption of nutrients and antioxidants and appears to be beneficial for asthma. although recent reviews have shown that zinc [ ] , garlic [ ] , echinacea purpurea [ ] or ginseng [ ] supplementation for several months may reduce cold incidence, there is insufficient evidence to recommend any vitamin or mineral supplementation in the management of asthmatic children without nutrient deficiency [ , ] . however, a large controlled trial showed echinacea was ineffective in reducing infection rate or symptom severity of hrv infection in healthy young adult volunteers [ ] . vitamin c supplementation failed to reduce the incidence of colds in the general population except in those exposed to short periods of extreme physical stress [ ] . finally, it is worth remembering that infants who are not breastfed have significantly higher risk of respiratory, gastrointestinal, and other infections, as breast milk is a biologically active substance containing antimicrobial and immunomodulatory elements [ ] [ ] [ ] . sleep and the circadian system exert a regulatory influence on immune functions. sleep deprivation can affect immune function in several ways including reduced natural killer cell activity, suppressed interleukin- production and increased levels of circulating proinflammatory cytokines [ , ] . there is also evidence for an enhanced susceptibility to the common cold and pneumonia with poor sleep efficiency [ , ] . air pollutants (nitrogen dioxide, ozone, particulate matter) and environmental tobacco smoke (ets) have long been correlated with multiple adverse effects on the immune system and susceptibility to viral respiratory tract infections in children [ ] [ ] [ ] [ ] . studies in europe and the united states have shown that % of children live with a smoker [ ] and they have approximately twice the risk of contracting a serious respiratory tract infection in early life [ ] . cigarette smoking leads to a longer duration of cough, greater frequency of abnormal auscultatory findings during acute respiratory tract illness [ , ] and higher risk for severe exacerbations [ ] . urinary leukotriene e levels identify children exposed to ets at high risk for asthma exacerbation [ ] . there is strong evidence that some pharmacological preparations can help prevent viral infection by specific effects on immune system. these results have been promising with a hope that using these strategies can attenuate the role of viruses in asthma inception. ancient physicians of the middle east prescribed yogurt for curing disorders of the stomach, intestines and for stimulation of appetite. it is written in the old persian testament that "abraham owed his longevity to the consumption of sour milk" [ ] . the popularity of probiotics and intestinal microbiota significantly increased when the nobel prize-winning russian scientist eli metchnikoff suggested in that the long life of bulgarian peasants resulted from their consumption of fermented milk products [ ] . the term probiotic, meaning for life, is used for live micro-organisms (typically of the bifidobacterium and lactobacillus species) administered in adequate amounts which confer a beneficial physiological effect on the host. prebiotics are nutrients, in particular oligosaccharides, which foster the growth of probiotics in the colon. the term synbiotics is used when a product contains both probiotics and prebiotics [ ] . up to trillion bacteria from different species colonize the human gut [ ] . this microbiota participates in: host metabolism, vitamin synthesis, control of epithelial cell growth, protection from infectious microbes, and helps proper development and function of the immune system. there is constant cross-talk between microbiota and gut-associated lymphoid tissue (the largest lymphoid tissue of the human body which contains more than % of all body lymphocytes) to establish mucosal immune tolerance in the gut. common mucosal immunity describes the phenomenon where immune cells, especially regulatory t-cells, traffic to and influence responses at other mucosal surfaces, including the lungs [ ] . alteration in the microbiota composition (dysbiosis) results in immunological dysregulation that may underlie many human diseases such as inflammatory diseases [ ] , obesity [ ] , allergy [ ] and autoimmunity [ ] . reduced bacterial diversity in the infant's gastrointestinal tract has been associated with an increased risk of allergic sensitization and allergic rhinitis but not asthma or atopic dermatitis [ ] . in the first year of life, especially the first few weeks, the microbiota of the newborn is highly variable during this critical time of post-natal maturation of the immune system. microbiota is shaped by genetic and environmental factors including: mode of delivery, neonates born by means of vaginal delivery are exposed to mothers gut, skin, and vaginal flora [ ] ; breast feeding and diet [ ] ; farm or urban living [ ] ; vitamin d status [ ] ; and antibiotic consumption [ , ] . this knowledge stimulated interest in the use of probiotics and prebiotics as the intentional introduction or encouragement of specific microbes to shape immune system development. specifically, the microbiota can activate distinct tolerogenic dendritic cells in the gut and through this interaction can drive regulatory t-cell differentiation that modulates both th and th responses inside and outside the gut [ ] [ ] [ ] . probiotics have been successfully used for the treatment of several gastrointestinal disorders (viral and antibiotic-associated diarrhea, inflammatory bowel disease) [ , ] . however, attempts to prevent or treat allergic disorders such as eczema, asthma and allergic rhinitis have had inconsistent results [ , , [ ] [ ] [ ] . there are a growing number of clinical trials using probiotics for the prevention and management of respiratory infections. while the precise mechanisms are largely unknown, speculations include: probiotics compete against pathogens; increase the barrier function in respiratory epithelium; immunostimulatory effects by enhancing cellular immunity with increased activity of natural killer cells and macrophages in airways [ ] . probiotics reduce the frequency of gastrointestinal and respiratory tract infections in children who attend day care centres [ ] . they have also been found to reduce the incidence of ventilator-associated pneumonia, respiratory infections in healthy and hospitalized children, and reduce the duration of common cold symptoms [ ] [ ] [ ] [ ] . one study demonstrated that that daily probiotic supplementation for six winter months in children three to five years of age reduced the incidence of fever, coughing and rhinorrhea by - % with no notable adverse events [ ] . probiotic combination with vitamins and minerals also reduced the duration and severity of common cold [ ] . a recent cochrane review of randomised controlled trials showed that probiotics were better than placebo in reducing the number of episodes of acute upper respiratory infections (uris) and reducing antibiotic use, while there were no differences in the mean duration of an episode and no increase in adverse events [ ] . probiotic foods such as probiotic milk or yogurt (functional foods) containing well-defined probiotic strains may reduce the risk of catching the common cold and represent a simple, safe, effective, available and affordable method for preventing respiratory infections in children [ , , [ ] [ ] [ ] [ ] [ ] [ ] . although there are several clinical trials that showed the preventive effect of probiotic, prebiotic [ ] or synbiotics treatments [ ] on respiratory infections, not all studies are positive with some failing to show any significant preventive effect [ ] . to explain the different results in clinical trials, it is of particular importance to point out that the immunomodulatory capabilities of probiotics are strain-dependent. difference in dosage, duration of intervention, population and environmental background may also affect the results. one major limitation in this field is that it is not possible to test just how "probiotic" a particular preparation is. technical advances will be required before some of the apparent discrepant results of studies can be resolved. several immunostimulants, including herbal extracts, bacterial extracts, synthetic compounds, have been promoted as increasing the immune defences of the respiratory tract. a recent cochrane review included data from placebo-controlled trials including participants below the age of years in which various types of "immunostimulants" were used to reduce acute respiratory tract infections, involving either upper or lower airways. the authors concluded that immunostimulants reduced the incidence of acute respiratory infections by % on average in susceptible children, but that trial quality was generally poor and a "high level of statistical heterogeneity was evident". a subgroup analysis focusing on bacterial immunostimulants, including om , produced similar results with lower statistical heterogeneity [ ] . om- bv (broncho-vaxom) is an immunostimulant extracted from eight common bacterial pathogens of the upper respiratory tract: haemophilus influenzae, diplococcus pneumoniae, klebsiella pneumoniae and ozaenae, staphylococcus aureus, streptococcus pyogenes and viridans, neisseria catarrhalis and has been used in several countries around the world for as long as years [ ] . recent studies showed that om- bv can reduce the number of acute respiratory infections by % to % compared with placebo in children with a history of recurrent infection [ ] . of particular interest, razi et al. showed that children between the age one and six years with recurrent wheezing who were given om- bv had a % reduction in the rates of wheezing over the subsequent months, compared to placebo (p < . ). in addition, the duration of each wheezing attack was two days shorter in the group given om- bv than in the group given placebo (p = . ) [ ] . again, direct evidence of the mechanisms involved are lacking from human studies. however, recent data from rodents shows that baseline regulatory t lymphocyte activity in the airways can be boosted by microbe-derived stimulation of the gut [ ] . bacterial immunostimulants were also shown to enhance innate immunity (i.e. intensification of phagocytosis) and adaptive immunity [ ] . as discussed above, evidence exists for an impaired innate immune response to respiratory viral infections in asthmatics [ ] . entry of rhinovirus into normal epithelial cells initiates a vigorous innate immune response with ifn-β secretion and apoptosis induction. in asthma, ifn-β and ifn-λ responses are impaired, resulting in viral replication, cell cytotoxicity, enhanced virion shedding and increased susceptibility to common cold [ , ] . epithelial cells of asthmatic patients responded to exogenous treatment with ifn-β exhibiting reduced rhinovirus release (cakebread, xu et al. ; jackson, sykes et al. ). if the proposed deficiency of type i and iii contribute to asthma exacerbations [ , , ] , correcting this deficiency with exogenous interferons would be a logical approach. the advantages of interferon application include the broad spectrum of activity with low risk of resistance development [ ] . prophylactic intranasal recombinant ifn-α and ifn-β have been shown to be effective against rhinovirus infection in humans [ ] [ ] [ ] . the results of these clinical trials are awaited with interest [ , ] . however, the systemic symptoms associated with severe viral infections, e.g. influenza, are associated with interferons, so careful dosing may be required. considering the occurrence of the local side effects, neutropenia and cost, the use of long-term prophylaxis with daily, intranasal administration of interferons is not feasible [ ] . however, randomized clinical trials using similar strategies are currently underway in adults with chronic respiratory disease and the results are keenly awaited. vitamin d deficiency is a common worldwide problem [ ] [ ] . beside importance for bone health, vitamin d plays an important role in adequate function of both the innate and adaptive immune systems including development of dendritic cells and regulatory t lymphocytes [ , ] production of antimicrobial proteins by airway epithelium [ ] , modifying the effect of intestinal flora on inflammatory disorders [ ] , and modulation of the inflammatory response to viral infections [ ] . recent reports suggest that vitamin d might play a role in the recent increase in allergic disease [ ] [ ] [ ] . vitamin d insufficiency has been associated with a higher incidence of respiratory tract infection, wheezing illness in children [ ] , reduced asthma control [ ] , emergency department visits, severe asthma exacerbations and hospitalizations [ , ] . in a recent study of children from five to years of age, with newly diagnosed asthma, vitamin d supplementation during the northern hemisphere winter months (september to july) prevented declining serum concentrations of (oh) d and reduced the risk of asthma exacerbation triggered by acute respiratory tract infections [ ] . macrolides possess anti-inflammatory and immunomodulatory properties extending beyond their antibacterial activity [ ] . indeed, they can attenuate pro-inflammatory cytokine production by bronchial epithelial cells, neutrophils and macrophages that may contribute to clinical improvement in many patients with chronic airway inflammation [ ] [ ] [ ] . azithromycin has anti-rhinoviral activity and can reduce hrv replication and release by increasing interferon production from epithelial cells [ , , ] . macrolide antibiotics inhibit rsv infection in human airway epithelial cells [ ] . a three weeks treatment with clarithromycin in rsv bronchiolitis had statistically significant effects on hospital length of stay and rate of readmission to the hospital within six months after discharge [ ] . however, direct evidence of macrolides preventing respiratory viral infection in children is lacking. as the majority of respiratory viral infections in young children are caused by hrv or rsv, we will briefly discuss anti-viral strategies to prevent hrv or rsv infections in asthmatic children. because there are more than serotypes of hrv, antiviral drugs are considered to be more effective than vaccination. antiviral agents have been designed to inhibit rhinovirus attachment, entry to the cell, viral uncoating, and rna and protein synthesis [ ] . table shows how intervention strategies can be targeted to various steps in the infective process. hrv has the icosahedrally shaped capsid formed by identical copies of viral capsid structural proteins vp - . the capsid protects the single-stranded, positive sense rna genome. while hrv-a and -b most often induce a self-limited upper respiratory infection, the recently discovered hrv-c was associated with slris in infants, bronchiolitis, and asthma exacerbations in children [ , ] . prevention of attachment, entry and uncoating hrv deposits on nasal or conjunctival mucosa and is transported to the posterior nasopharynx by mucociliary action of epithelial cells [ ] . the so-called major group of hrv uses intercellular adhesion molecule- (icam- ) as their receptor [ ] and the minor group attach to low density lipoprotein (ldl) receptor and very-ldl (vldl) receptors on epithelial cells in the adenoid area to bind and enter cells [ , ] . viral attachment can be prevented by specific anti-hrv neutralizing antibodies, anti-receptor antibodies and soluble receptor molecules. endothelial cells express histamine receptors and increased adhesion molecule expression, such as icam- , was demonstrated by histamine infusion. second-generation h -antihistamines decrease expression of icam- on cultured bronchial epithelial cells [ ] . zinc may also act as an antiviral agent by reducing icam- levels [ ] . the monoclonal antibody to the cellular icam- was not effective. cfy (coldsol) is a nasal spray multivalent fab fusion proteins against icam- with a better avidity and in vitro potency against hrv [ ] . tremacamra, a soluble intercellular adhesion molecule reduced the severity of experimental rhinovirus infection [ ] . pleconaril, an orally administered antiviral drug, acts by binding to a hydrophobic pocket in viral protein , and stabilizes the protein capsid so that the virus cannot release its rna genome into the target cell. outcomes of clinical trials with pleconaril have revealed mixed results and new compounds are currently being developed [ ] . despite extensive research, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. ruprintrivir selectively inhibits hrv c protease and shows potent, broad-spectrum anti-hrv activity in vitro. ruprintrivir nasal spray ( % solution) prophylaxis reduced the proportion of subjects with positive viral culture by % and reduce viral titers, but did not decrease the frequency of colds [ ] . hrv rna synthesis during replication can be blocked by deoxyribozymes [ ] , morpholino oligomers [ ] , and small interfering ribonucleic acids [ ] . the novel antiviral therapies that have been discovered recently, may one day add significantly to the armamentarium of antiviral agents, against respiratory viral infections in asthmatic children. maternally-derived rsv neutralizing antibodies help to protect infants against rsv hospitalization [ ] . palivizumab, a humanised monoclonal antibody against the rsv fusion protein is effective against rsv and wheezing in children and reduces hospitalization in high-risk individuals [ , ] . rsv prophylaxis with palivizumab significantly reduced the relative risk of subsequent recurrent wheezing in nonatopic premature infants [ ] . motavizumab is another monoclonal antibody against rsv, with an approximately -fold increase in ability to neutralize rsv and fold increase in ability to reduce viral titers compared to palivizumab [ , ] . motavizumab was also found to be superior to palivizumab in reducing outpatient medically attended lower respiratory illness by % [ ] . vaccination against hrv and rsv have been in development for quite some time, but there are no safe and effective vaccines at present [ , ] . high rates of exposure to viruses in early life, presence of more than serotypes of hrv, the presence of maternal antibodies, the risk of vaccine induced disease and relative immaturity of the infant immune system make effective vaccination difficult [ , , ] . respiratory viral infections are major contributors to the global burden imposed by asthma. in early life, they contribute to the inception of asthma and are responsible for most of the acute exacerbations for asthma in childhood. while the debate is not completely settled, children at high risk of developing asthma and those with established asthma may be at increased risk of acquiring respiratory viral infections and may be less able to contain these to the upper airway. several simple general strategies can be used to help prevent respiratory viral infections in asthmatic children (table ) , with good personal hygiene, hand-washing and avoidance of cigarette smoke likely to reduce respiratory viral infections. general immuno-stimulatory strategies, such as eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, e.g. om- , may reduce recurrent infections in susceptible children. while research continues on specific anti-viral therapies, including vaccination, there are no currently available practical therapies that are suitable for widespread use. the role of preventative strategies in primary prevention of asthma in high risk children is of considerable academic interest and a number of studies are currently in the pipeline. the results are awaited with interest. the authors declare they have no competing interests. author's contribution ha and pds conceived and designed the review. all authors reviewed the articles, abstracted data, and participated in the data synthesis. ha, pds, ysc drafted the current manuscript, with critical review by pds and cmj. all authors contributed, read and approved the final manuscript. the global burden of asthma: executive summary of the gina dissemination committee report respiratory syncytial virus in early life and risk of wheeze and allergy by age years respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma do early-life viral infections cause asthma? rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in high-risk children respiratory viruses and exacerbations of asthma in adults how viral infections cause exacerbation of airway diseases persistence of rhinovirus rna after asthma exacerbation in children study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma immune pathways for translating viral infection into chronic airway disease host epithelial-viral interactions as cause and cure for asthma the epithelium takes centre stage in asthma and atopic dermatitis the sentinel role of the airway epithelium in asthma pathogenesis sensing the outside world: tslp regulates barrier immunity lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient type iii interferon-lambda production in asthma exacerbations counterregulation between the fcepsilonri pathway and antiviral responses in human plasmacytoid dendritic cells tlr polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide interaction between adaptive and innate immune pathways in the pathogenesis of atopic asthma: operation of a lung/ bone marrow axis antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children asthma exacerbations: origin, effect, and prevention severe exacerbations and decline in lung function in asthma relationship between increased airway responsiveness and asthma severity in the childhood asthma management program understanding the september asthma epidemic predicting asthma exacerbations in children role of viral respiratory infections in asthma and asthma exacerbations the role of viruses in acute exacerbations of asthma asthma exacerbations: origin, effect, and prevention the september epidemic of asthma exacerbations in children: a search for etiology viruses and asthma evidence for a causal relationship between respiratory syncytial virus infection and asthma rhinovirus and the initiation of asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma palivizumab long-term respiratory outcomes study g: the effect of respiratory syncytial virus on subsequent recurrent wheezing in atopic and nonatopic children contemporaneous maturation of immunologic and respiratory functions during early childhood: implications for development of asthma prevention strategies the role of rhinovirus infections in the development of early childhood asthma the emerging relationship between the airway microbiota and chronic respiratory disease: clinical implications microbes and asthma: the missing cellular and molecular links association of bacteria and viruses with wheezy episodes in young children: prospective birth cohort study incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients the human rhinovirus: human-pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections persistence of rhinovirus and enterovirus rna after acute respiratory illness in children the common cold and decongestant therapy physical interventions to interrupt or reduce the spread of respiratory viruses back to the future: rising to the semmelweis challenge in hand hygiene effect of handwashing on child health: a randomised controlled trial illness transmission in the home: a possible role for alcohol-based hand gels a randomized, controlled trial of a multifaceted intervention including alcohol-based hand sanitizer and hand-hygiene education to reduce illness transmission in the home scheduled hand washing in an elementary school population effectiveness of a training program in reducing infections in toddlers attending day care centers effect of infection control measures on the frequency of upper respiratory infection in child care: a randomized, controlled trial effects of hand hygiene campaigns on incidence of laboratoryconfirmed influenza and absenteeism in schoolchildren a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection. clinical infectious diseases: an official publication of the infectious diseases society of america face masks to prevent transmission of influenza virus: a systematic review the antiinflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease moderate to vigorous physical activity and risk of upper-respiratory tract infection upper respiratory tract infection is reduced in physically fit and active adults mucosal iga and urti in american college football players: a year longitudinal study physical activity and exercise in asthma: relevance to etiology and treatment respiratory infection risk in athletes: association with antigen-stimulated il- production and salivary iga secretion meditation or exercise for preventing acute respiratory infection: a randomized controlled trial serum vitamin d levels and markers of severity of childhood asthma in costa rica serum vitamin d levels and severe asthma exacerbations in the childhood asthma management program study influence of dietary components on regulatory t cells zinc for the common cold garlic for the common cold echinacea for preventing and treating the common cold north american (panax quinquefolius) and asian ginseng (panax ginseng) preparations for prevention of the common cold in healthy adults: a systematic review a medical nutrition therapy primer for childhood asthma: current and emerging perspectives summary of the bts/sign british guideline on the management of asthma an evaluation of echinacea angustifolia in experimental rhinovirus infections vitamin c for preventing and treating the common cold breastfeeding: maintaining an irreplaceable immunological resource breastfeeding and otitis media: a review of recent evidence diet for the prevention of asthma and allergies in early childhood: much ado about something? sleep and immune function effects of sleep and circadian rhythm on the human immune system sleep habits and susceptibility to the common cold a prospective study of sleep duration and pneumonia risk in women second hand smoke exposure in children: environmental factors, physiological effects, and interventions within pediatrics smoking and the outcome of infection air pollution and respiratory viral infection the response of children with asthma to ambient particulate is modified by tobacco smoke exposure meta-analysis on the association between environmental tobacco smoke (ets) exposure and the prevalence of lower respiratory tract infection in early childhood association between cigarette smoking and acute respiratory tract illness in young adults the relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking urinary leukotriene e levels identify children with tobacco smoke exposure at risk for asthma exacerbation probiotics: what are they? what are their effects on gut physiology? metchnikoff and the centenary of probiotics: an update of their use in gastroenteric pathology during the age of development microbial manipulation of immune function for asthma prevention: inferences from clinical trials current concepts of the intestinal microbiota and the pathogenesis of infection gershwin me: probiotics and immunity probiotics for the treatment of allergic rhinitis and asthma: systematic review of randomized controlled trials a core gut microbiome in obese and lean twins reduced diversity of the intestinal microbiota during infancy is associated with increased risk of allergic disease at school age the gut microbiota shapes intestinal immune responses during health and disease factors influencing the composition of the intestinal microbiota in early infancy establishment of the gut microbiota in western infants exposure to environmental microorganisms and childhood asthma gut microbiota, probiotics, and vitamin d: interrelated exposures influencing allergy, asthma, and obesity? use of antibacterials in infancy: clinical implications for childhood asthma and allergies probiotics and lung diseases influence of gastrointestinal commensal bacteria on the immune responses that mediate allergy and asthma influence of dietary components on regulatory t cells a comparison between traditional yogurt and probiotic yogurt in noninflammatory acute gastroenteritis probiotics for the prevention of pediatric antibiotic-associated diarrhea a differential effect of probiotics in the prevention of eczema and atopy: a double-blind, randomized, placebo-controlled trial effect of a new synbiotic mixture on atopic dermatitis in children: a randomized-controlled trial probiotics for allergic diseases: realities and myths effect of intranasal administration of lactobacillus pentosus s-pt on influenza virus infection in mice lactobacillus gg in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers: a randomized, double-blind, placebo-controlled trial probiotic prophylaxis of ventilatorassociated pneumonia a blinded, randomized, controlled trial effect of long term consumption of probiotic milk on infections in children attending day care centres: double blind, randomised trial lactobacillus gg in the prevention of nosocomial gastrointestinal and respiratory tract infections probiotic bacteria reduced duration and severity but not the incidence of common cold episodes in a double blind, randomized probiotic effects on cold and influenza-like symptom incidence and duration in children effect of a dietary supplement containing probiotic bacteria plus vitamins and minerals on common cold infections and cellular immune parameters probiotics for preventing acute upper respiratory tract infections reducing the risk of infection in the elderly by dietary intake of yoghurt fermented with lactobacillus delbrueckii ssp. bulgaricus oll r- immunologic effects of yogurt use of a fermented dairy probiotic drink containing lactobacillus casei (dn- ) to decrease the rate of illness in kids: the drink study. a patient-oriented, double-blind, cluster-randomized, placebo-controlled, clinical trial effect of long-term continuous consumption of fermented milk containing probiotic bacteria on mucosal immunity and the activity of peritoneal macrophages influence of nutritional knowledge on perceived healthiness and willingness to try functional foods taxonomy and important features of probiotic microorganisms in food and nutrition prebiotics, immune function, infection and inflammation: a review of the evidence long-term safety and impact on infection rates of postnatal probiotic and prebiotic (synbiotic) treatment: randomized, double-blind, placebo-controlled trial efficacy of probiotic lactobacillus gg on allergic sensitization and asthma in infants at risk immunostimulants for preventing respiratory tract infection in children can we prevent exacerbations of asthma caused by common cold viruses? om- bv, an immunostimulant in pediatric recurrent respiratory tract infections: a systematic review the immunostimulant om- bv prevents wheezing attacks in preschool children boosting airway t-regulatory cells by gastrointestinal stimulation as a strategy for asthma control bacterial immunostimulantsmechanism of action and clinical application in respiratory diseases epithelium dysfunction in asthma role of rhinovirus infections in asthma il- a (ifn-lambda ) modulates lung dc function to promote th immune skewing and suppress allergic airway disease intranasal interferon-alpha- for prevention of natural rhinovirus colds interferon-beta-ser as prophylaxis against experimental rhinovirus infection in volunteers an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of huifn-alpha- a mechanistic role for type iii interferon-lambda in asthma exacerbations mediated by human rhinoviruses interferon-lambda as a new approach for treatment of allergic asthma? vitamin d deficiency high prevalence of vitamin d inadequacy and implications for health vitamin d and asthma vitamin d recommendations: beyond deficiency induction of cathelicidin in normal and cf bronchial epithelial cells by , -dihydroxyvitamin d( ) respiratory epithelial cells convert inactive vitamin d to its active form: potential effects on host defense potential mechanisms for the hypothesized link between sunshine, vitamin d, and food allergy in children regional differences in epipen prescriptions in the united states: the potential role of vitamin d prevalence of eczema and food allergy is associated with latitude in australia vitamin d, respiratory infections, and asthma relationship between serum -hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey low serum -hydroxyvitamin d levels are associated with increased risk of viral coinfections in wheezing children vitamin d supplementation in children may prevent asthma exacerbation triggered by acute respiratory infection macrolide antibiotics in the treatment of asthma. an update anti-inflammatory effects of macrolides in lung disease macrolides in the treatment of asthma azithromycin inhibits il- production of t helper type cells from asthmatic children macrolide antibiotics and asthma treatment azithromycin induces anti-viral responses in bronchial epithelial cells macrolide antibiotics inhibit respiratory syncytial virus infection in human airway epithelial cells clarithromycin in the treatment of rsv bronchiolitis: a double-blind, randomised, placebo-controlled trial a novel group of rhinoviruses is associated with asthma hospitalizations novel human rhinoviruses and exacerbation of asthma in children incubation periods of experimental rhinovirus infection and illness. clinical infectious diseases: an official publication of the infectious diseases society of america a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus multiple receptors involved in human rhinovirus attachment to live cells levocetirizine inhibits rhinovirus-induced icam- and cytokine expression and viral replication in airway epithelial cells duration and severity of symptoms and levels of plasma interleukin- receptor antagonist, soluble tumor necrosis factor receptor, and adhesion molecules in patients with common cold treated with zinc acetate viral receptor blockage by multivalent recombinant antibody fusion proteins: inhibiting human rhinovirus (hrv) infection with cfy efficacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection -a randomized clinical trial combating enterovirus replication: stateof-the-art on antiviral research phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray -percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers gaining target access for deoxyribozymes a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism respiratory syncytial virus neutralizing antibodies in cord blood, respiratory syncytial virus hospitalization, and recurrent wheeze understanding the mechanisms of viral induced asthma: new therapeutic directions palivizumab prophylaxis, respiratory syncytial virus, and subsequent recurrent wheezing development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract a review of palivizumab and emerging therapies for respiratory syncytial virus motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial vaccination for asthma exacerbations viral respiratory tract infections and asthma: the course ahead association between human rhinovirus c and severity of acute asthma in children submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors wish to acknowledge dr. catherine gangell for reviewing the manuscript. key: cord- -deuytbml authors: maffey, alberto f.; barrero, paola r.; venialgo, carolina; fernández, francisco; fuse, valentina a.; saia, mariana; villalba, analía; rodríguez fermepin, marcelo; teper, alejandro m.; mistchenko, alicia s. title: viruses and atypical bacteria associated with asthma exacerbations in hospitalized children date: - - journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: deuytbml objectives and working hypothesis: to evaluate the prevalence of respiratory viruses mycoplasma pneumoniae and chlamydophila pneumoniae and gain insight into their seasonal circulation pattern in children with acute asthma exacerbations in a temperate southern hemisphere region. study design: patients hospitalized between months and years of age were included in a ‐year prospective, observational, cross‐sectional study. respiratory secretions were collected and the presence of different viruses and atypical bacteria analyzed by immunofluorescence and polymerase chain reaction. results: two hundred nine patients ( females) aged (mean ± sd) . ± years were included. a potential causative agent was detected in % of the patients. the most frequently detected viruses were respiratory syncytial virus (hrsv) (n = ; %) and rhinovirus (hrv) (n = ; . %); m. pneumoniae and c. pneumoniae were detected in . % and % of the cases, respectively. patients with hrsv (vs. hrv) were hospitalized for a longer time ( . vs. . days, p = . ), required more days of oxygen supply ( . vs. . , p = . ), had a longer duration of the exacerbation before hospitalization ( . vs. . days, p = . ) and were younger ( . vs. . years, p = . ). three peaks of admissions were observed. a first peak (early autumn) caused by hrv, a second peak (winter) caused mainly by hrsv and a third one (spring), caused by hrsv, an increase in hmpv together with a second outbreak of hrv. conclusions: children with an acute asthma exacerbation presented a high prevalence of respiratory viruses. most hospitalizations corresponded to seasonal increases in prevalence of hrv and hrsv. pediatr pulmonol. ; : – . © wiley‐liss, inc. asthma is the most-frequent pediatric chronic disease, and its prevalence is increasing in numerous regions of the world. the international study of asthma and allergies in childhood (isaac) has reported that in argentina the current prevalence of asthma is . % in children aged years old and . % in children aged . respiratory exacerbations are the main source of morbidity, mortality, school absence, and health expenses associated with asthma. infections associated with respiratory viruses and atypical bacteria are the main cause of asthma exacerbation. about - % of the wheezing episodes in infants and - % in children and adolescents are triggered by respiratory viruses. various viral agents, including the respiratory syncytial virus (hrsv), influenza a and b (fluav and flubv), parainfluenza (hpiv), adenovirus (hadv), rhinovirus (hrv), enterovirus (hev), coronavirus (hcov), and the recently described metapneumovirus (hmpv) and bocavirus (hbov) have been detected in patients presenting with asthma exacerbation. while hrsv is the main agent associated with wheezing in infants and preschool children hrv is the most-frequent agent detected in school children, adolescents, and adults. infections caused by atypical bacteria (mycoplasma pneumoniae and chlamydophila pneumoniae) have been associated not only with asthmatic exacerbations but also with the development of chronic infections that may contribute to the persistence and severity of asthma. , numerous adequately treated asthmatic patients continue to experience wheezing episodes, so studies are necessary to understand the relationship between the inflammatory process triggered by various infectious agents and the events that lead to the loss of asthma control. , this knowledge will assist the development of prevention and treatment strategies to help decrease the morbidity caused by bronchial asthma. [ ] [ ] we performed a cross-sectional study to determine by means of immunofluorescence assay (ifa) and molecular techniques the prevalence of traditional and newly described respiratory viruses and atypical bacteria in children hospitalized due to an acute asthma exacerbation in buenos aires, a temperate southern hemisphere city. the study was performed at the ricardo gutiérrez children's hospital in buenos aires between january and december , . the study protocol was approved by the institutional review board of the hospital. legally authorized representatives of the children provided informed consent. patients included in the study were between months and years of age, with a history of two or more previous wheezing episodes diagnosed by a physician and presenting with a new episode severe enough to require hospitalization. forty-two ( %) patients were under months, an age group where it is difficult to establish a definite diagnosis of asthma. because of the difficulty in differentiation of asthma from wheezing episodes of other origin in very young children, to be included in the study patients under years of age needed to have an index for the prediction of asthma according to the criteria of castro-rodríguez et al. children over three needed to comply with the diagnostic criteria of bronchial asthma according to the gina guidelines. the exclusion criteria were premature birth and the presence of a chronic disease of the pulmonary or cardiovascular system, metabolic disorders, immunosupression, genetic or neurological disorders. we recorded number of weeks of gestation and weight at birth, familial history of asthma, allergic rhinitis or atopic eczema in first degree relatives, passive smoking, medications used to achieve asthma control, age at first wheezing episode, and hospitalizations in the last months by interviewing the parents and reviewing medical records. during hospitalization, data on clinical evolution (days of hospitalization and oxygen requirement), treatment required (bronchodilators, systemic corticosteroids, antibiotics, mechanical respiratory assistance), and complications presented (pneumonia and atelectasis) were recorded. the interval of time (days) between the onset of the respiratory exacerbation and admission and time between admission and the collection of the respiratory specimen were also recorded. the results of the characterization of the respiratory viruses studied (hrsv, hadv, fluav and flubv, hpiv- , , and , hmpv, hrv, hev, hcov, hbov) as well as of the atypical bacteria m. pneumoniae and c. pneumoniae were registered. due to the wide range of age of the patients included, for statistical analysis of the different variables the patients were arbitrarily divided into three groups: children under year of age, children between and years of age, and children between and years of age. a nasopharyngeal aspirate (bsn , laboratorio barcat, buenos aires, argentina) was obtained from each patient on admission. on patients in whom respiratory secretions could not be collected by this method, a nasal swab (transport swab , copan, brescia, italy) was obtained. the sample was kept at c until submission to the laboratory. the respiratory sample was processed the day it was collected by the ifa to detect hrsv, hadv, hpiv- , , and , fluav, and flubv (light diagnostics, chemicon int., temecula, ca). a total of , cells per ml were considered adequate for ifa detection. the secretions were kept at À c for further studies with polymerase chain reaction (pcr) and reverse transcription pcr (gotaq , promega, madison, wi; onestep rt-pcr, qiagen, valencia, ca). the viruses (hrsv, fluav, flubv, hpiv, hadv, hrv, hev, hcov, hmpv, and hbov) and atypical bacteria (m. pneumoniae and c. pneumoniae) were determined in separate reactions according to the protocols previously described. a positive case was defined as that in which at least one infectious agent was detected. descriptive statistics were performed using epi . for comparison of groups data were imported into r statistical programming language environment, version . . (available from http://www.r-project.org). variables examined to compare patients with single agents versus co-detections and between hrsv and hrv single detections were age, gender, passive smoking, familial history of asthma or atopy, duration of exacerbation before hospitalization, days on oxygen supply, length of hospitalization and complications (atelectasis and pneumonia). chi-square and fisher's exact tests were used for comparison of categorical variables. for numerical variables we used student's t-test or wilcoxon test when it was not possible to assume a normal distribution. the values are expressed as mean ae standard deviation. the probability level to determine statistical significance was . . a total of patients that fulfilled the inclusion criteria were admitted during the study period. five patients, all less than years of age, were excluded once studies were initiated to rule out differential diagnosis of asthma. two patients declined to participate, and parents of another patient were not present to give the informed consent. a total of ( %) out of the patients studied were under year of age, ( %) between and years of age, and ( %) between and years of age. table shows the demographic and clinical characteristics and complications present in the population studied. additional information on the first wheezing episode was obtained on patients: ( %) had their first wheezing episode in the first years of life and % of these wheezed in their first year. the interval of time between the onset of the respiratory exacerbation and admission was found to be (mean ae sd): . ae . days, between the onset of the respiratory exacerbation and the collection of the respiratory specimen . ae . days, and between admission and the specimen collection . ae . days. a total of ( %) patients had been prescribed an asthma controller therapy, but only ( %) were complying with it adequately: ( %) of the children under year of age, ( %) of the children between and years of age, and ( %) of the children between and years of age. sixteen ( %) of those who were complying with therapy received budesonide, ( %) fluticasone, ( %) fluticasone plus salmeterol, and ( %) montelukast. in the physical exam carried out at admission, ( %) of the patients presented cough, ( %) rhinorrhea, ( %) fever ! c, ( %) pharyngitis, and ( %) conjunctivitis. in addition, ( . %) were diagnosed with acute otitis media. overall, ( %) were found to have an upper respiratory infection. during hospitalization, all the patients received oxygen treatment through a nasal cannula or a facemask, shortacting bronchodilators (nebulized albuterol) and systemic corticosteroids (hydrocortisone, dexametasone, or methyl-prednisolone); but none of them required mechanical ventilation. there were no significant differences when evaluating the presence of complications (atelectasis and pneumonia) when an infectious agent was detected or when codetections occurred (p ¼ . and p ¼ . , respectively). samples of respiratory secretions were obtained through a nasopharyngeal aspirate from ( %) patients. in ( %) patients respiratory secretions could not be obtained by this method, so a nasal swab was pediatric pulmonology table ) . patients with co-detections were significantly younger than those without co-detections ( . vs. . years of age; p ¼ . ). no hpiv- was detected. the four cases of hcov and the only case of flubv were present in co-detections. the importance of the two more-prevalent respiratory viruses (hrsv and hrv) as single detection, as well as that of the viral co-detections, was analyzed related to the main variables studied. we found that the patients with hrsv were hospitalized later in the course of their disease ( . vs. . days, p ¼ . ), experienced a longer hospitalization ( . vs. . days, p ¼ . ), required more days of oxygen supplementation ( the number of hospitalizations varied seasonally and was associated with the circulation of the more frequently isolated viruses (fig. ) . in the present study, a potential causative agent of an acute asthma exacerbation was identified in % of the patients studied. this rate of detection is in agreement with those reported in the literature, which vary between % and % of positive cases according to the age of the population studied, the number of infectious agents evaluated, and the diagnosis techniques used. , the pcr technique allowed the detection of hrv, hev, hmpv, hbov, hcov, m. pneumoniae, and c. pneumoniae, and also contributed to the identification of cases that had not been detected by ifa. however, it should be pointed out that while the ifa technique detects viral antigens in patients that present an active viral infection, the higher sensitivity of the pcr technique allows the detection of nucleic acids that may correspond either to a current infection or to a past infection. in effect, nokso-koivisto et al. described the presence of positive picornavirus rna in respiratory samples obtained from children without concurrent respiratory symptoms. in our study, % of the patients had an active upper respiratory infection at the time of hospital admission, thereby lowering the risk of diagnosing false positive cases. the most frequently detected agent was hrsv, in both the group of children between and years of age and the group of children between and years of age. this finding supports previous observations that hrsv can cause acute exacerbations of chronic diseases at any age. community-based studies in school children have found that hrv is the most prevalent virus, responsible for up to % of the asthma exacerbations. in our study, we detected hrv in % of the patients. this difference could be that our patients were not only hospitalized, but also younger. some concerns could be regarded about the sample size that could lack sufficient statistical power, specially in smaller subgroups of analysis. however, several differences between groups reached statistical significance. patients with exacerbations caused by hrv had a significantly shorter number of days from the beginning of the respiratory symptoms to hospital admission, and also shorter hospitalization and less oxygen requirement days than those with exacerbations caused by hrsv. although the children affected by hrv were older and thus could be expected to have a wider airway caliber, this difference may be because the exacerbations triggered by hrv show a better response to treatment than those triggered by other respiratory viruses due to different pathogenic mechanisms of airway obstruction. thus, it was not possible to determine if the difference in clinical outcome observed might be related to the age of the patients' group or to the different pathogenic mechanisms of hrv and hrsv. in our cohorting, other common respiratory viruses, such as fluv, hbov, hcov, hmpv, and hpiv, exhibited a much lower prevalence than hrsv and hrv. it is well known that fluv triggers asthma exacerbations less frequently than other respiratory viruses. due to its frequent presence in co-detection and its long period of excretion after an active infection, the role of hbov as a causative agent of asthmatic exacerbations requires further studies. the detection of hmpv, hcov, hpiv, and hadv, while higher in infants and school children, was significantly lower than that of hrsv and hrv. in asthmatic hospitalized children, studies have reported prevalence of % for m. pneumoniae and - % for c. pneumoniae, which is higher than that found by our group ( . % for m. pneumoniae and % for c. pneumoniae). this difference may be due to a lower circulation of these agents during the year in which the study was performed or to a lower detection rate, as we did not use serological methods. another possible explanation could be the preponderance of young children sampled, as m. pneumoniae is usually less frequent under years of age than in school age children. therefore, treatment with macrolides is not justified in all patients presenting an asthma exacerbation but should be considered in children presenting persistent symptoms that cannot be controlled with conventional treatment. in almost a quarter of patients ( %) we found evidence of a co-detection between respiratory viruses and/or atypical bacteria. we did not find significant differences when analyzing the occurrence of co-detections between the different agents involved and the hospitalization or pediatric pulmonology oxygen requirement days and the development of complications. , however, the presence of such a high index of co-detection points to the need to revise the concept of ''cohortization'' or grouping patients in hospital rooms. co-detections in which any of the viral agents recently described is involved can favor the development of nosocomial infections. although previously described in other regions of the world, to our best knowledge, this is the first report of respiratory viruses and atypical bacteria associated to wheezing in children described in the southern cone of america. in the near future, when antiviral therapies become more readily available, to know the regional patterns of circulation of viral agents might be useful to treat specific viral respiratory infections, helping to prevent asthmatic exacerbations. [ ] [ ] in conclusion, the present study confirms the high prevalence of respiratory viruses in hospitalized children with an acute asthma exacerbation, highlights the importance both of hrsv and hrv in all age groups, and describes their seasonal pattern in a temperate southern hemisphere location. the asthma epidemic prevalence of asthma symptoms in latin america: the international study of asthma and allergies in childhood (isaac) respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children human metapneumovirus and acute wheezing in children human bocavirus and acute wheezing in children increased frequency of detection of chlamydophila pneumoniae in asthma detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children new treatment regimes for virus-induced exacerbations of asthma viruses and asthma, inception, exacerbation, and possible prevention the role of viral infections in the natural history of asthma early identification of atopy in the prediction of persistent asthma in children definition, assessment and treatment of wheezing disorders in preschool children: an evidence-based approach a clinical index to define risk of asthma in young children with recurrent wheezing global strategy for asthma management and prevention: gina executive summary new respiratory viruses in children months to years old with recurrent wheeze prevalence of viral respiratory tract infections in children with asthma role of viruses and atypical bacteria in exacerbations of asthma in hospitalized children: a prospective study in the nord-pas de calais region (france) comparison of real-time pcr assays with fluorescentantibody for diagnosis of respiratory virus infections in children human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms respiratory syncytial virus infection in elderly and high-risk adults community study of role of viral infections in exacerbations of asthma in - year old children prednisolone reduces recurrent wheezing alter a first wheezing episode associated with rhinovirus infection or eczema influenza virus and acute asthma in children respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children importance of acute mycoplasma pneumoniae and chlamydia pneumoniae infections in children with wheezing the effect of telithromycin in acute exacerbations of asthma impact of human metapneumovirus and respiratory syncytial virus co-infection in severe bronchiolitis absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection a comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis and the implications for a cohorting strategy respiratory viruses seasonality in children under five years of age in buenos aires, argentina. a five-years analisis managing childhood asthma: challenge of preventing exacerbations the authors thank dr. a. sonia buist and the staff of the ats methods in epidemiologic, clinical and operations research (mecor) for encouraging our research, and very specially dr. shannon carson for kindly correcting the manuscript. also, we would like to thank mrs. patricia riveiro, bioq. m. angeles marques, and bioq. andrea c. entrocassi for their skillful assistance in diagnostic procedures. we are indebted to dr. van der hoek for kindly providing the positive control to detect coronavirus. key: cord- -rrf dbnb authors: wood, lisa g.; powell, heather; grissell, terry v.; davies, bronwyn; shafren, darren r.; whitehead, bruce f.; hensley, michael j.; gibson, peter g. title: persistence of rhinovirus rna and ip‐ gene expression after acute asthma date: - - journal: respirology doi: . /j. - . . .x sha: doc_id: cord_uid: rrf dbnb background and objective: viral nucleic acid may be detected for up to months after an acute asthma deterioration, but the pattern and consequences of viral persistence after acute asthma are incompletely understood. this study investigates the frequency of viral persistence after acute asthma, assesses viral infectivity and determines the host inflammatory responses to viral persistence. methods: adults and children presenting to hospital with acute asthma and a confirmed respiratory virus infection were studied acutely and at recovery – weeks later by clinical evaluation and induced sputum for viral and inflammatory mediator detection. results: viral rna was detected during both acute asthma and recovery visits in subjects (viral persistence), whereas in subjects viral rna had cleared by recovery (viral clearance). the following viruses were detected at recovery: human rhinovirus: ; respiratory syncytial virus: ; influenza: . in subjects with viral persistence, eight isolates were different to the virus detected at visit . forty‐four per cent of the human rhinovirus isolates were infective at recovery. asthma and infection severity were similar in the viral clearance and viral persistence groups. viral persistence was associated with elevated il‐ mrna and inducible protein‐ gene expression. conclusions: respiratory viral detection after acute asthma is common, and most often persistence is with non‐infective human rhinovirus. there is a host inflammatory response with an altered cytokine environment, and the viral rna can be source of persistent infection. these effects may have longer‐term consequences in asthma. asthma exacerbation is a significant problem for adults and children. respiratory viral infections, in particular human rhinovirus (hrv) infection, represent the main causal factor. an innate immune defect in asthmatic epithelium can result in enhanced virus replication in virally induced asthma. , hrv, an rna virus, is readily degraded by rnase activity and, consequently, shedding of hrv is typically expected to occur only briefly during a viral infection. the impaired interferon response in asthmatic epithelium that has been observed in vitro could result in delayed viral clearance, and several reports have documented persistence of hrv nucleic acid in - % of children, at weeks after presentation to the emergency department with acute asthma or wheezing. several studies have also reported detection of hrv nucleic acid in up to % of asymptomatic subjects. [ ] [ ] [ ] the unexpected persistence of viral rna in the upper respiratory tract of asthmatic children indicates a possible connection between viral infection and recurrent asthma. the clinical implications of these findings are unknown; however, airway hyperresponsiveness persists after virus infection in asthmatic children. there are several possible explanations for the persistence of hrv after acute asthma with viral infection. these include: • residual infection with the same viral serotype. • the virus is non-infectious, and represents nucleic acid remnants, or viral quasispecies , representing viral mutations. • re-infection with a different virus or viral serotype. • the virus is colonizing, and not eliciting a host response. the consequences of viral persistence could include enhancing chronic inflammation, or changing the pattern of local cytokine or chemokine responses. in rsv infection, latent or persistent viral infection has been linked to adverse asthma outcomes in the long term, and similar results have been reported following hrv infection. thus viral persistence could have long-term adverse effects. this study sought to address the pattern and consequences of viral persistence after acute asthma exacerbation by investigating the frequency of viral persistence following acute asthma, assessing the infectivity of the persistent virus, and determining the host inflammatory and cytokine response in asthmatic subjects with viral persistence. the subjects with acute asthma were a subset of a previously described cohort who were selected based on: age > years; presentation to john hunter hospital emergency department with acute asthma; virus detected by pcr and/or culture; and attendance for a follow-up visit. the study was approved by the hunter new england research ethics committee and written informed consent obtained. subjects were assessed on two occasions, within h of hospital admission and - weeks later. during hospital admission, subjects were managed according to hospital guidelines and discharged when stable. at both visits subjects completed asthma history and asthma control questionnaires, provided nasal and throat swabs and underwent spirometry combined with sputum induction using ultrasonic nebulization of isotonic saline as previously described. selected portions of induced sputum were allocated to: (i) a lytic solution (buffer rlt; qiagen, hilden, germany) for rna extraction and subsequent mrna expression analysis and virus pcr; (ii) in vitro culture in viruspermissive human epithelial cell lines (hela and hep- ) for outgrowth of respiratory viruses; and (iii) sequential dithiothreitol and pbs for cellular dispersion and profiling and storage of cell free supernatant as previously described. briefly, cytospins were prepared, stained (may-grunwald geimsa) and a differential cell count obtained from non-squamous cells. the remaining solution was centrifuged ( g, min, °c) and the cell-free supernatant was aspirated and stored at - °c. rna was extracted (rneasy rna mini kit, qiagen) and reverse transcription performed using random hexamers and superscript ii rnase h+ reverse transcriptase enzyme as previously described. samples were assayed for the presence of rhinovirus (hrv), enterovirus (hev), influenza virus types a and b (ifa, ifb), respiratory syncytial virus types a and b (rsva, rsvb), metapneumovirus (mpv) and non-sars coronavirus (cov) virus rna transcripts using real-time 'taqman' methodology pcr assays (table ) . a fragment of approximately nucleotides encompassing the vp /vp region and the hypervariable region in the ′-non-coding region was amplified with one-step rt-pcr using hrv specific primers. rt-pcr products were visualized, purified (qiaquick gel extraction kit, qiagen), and nucleotide sequences determined in cycle sequencing reactions using dyenamic et dye terminator cycle sequencing kit (megabace) (ge healthcare, fairfield, ct, usa). sequence data were analysed (sequencher, version . . ) and nucleotide-nucleotide alignments performed using ncbi blast for assessment of percentage identity with prototype strains (genbank). taqman primers and probes specific for il- , il- , ip- , mip- a, and rantes mrna were combined with those for the housekeeping gene human s ribosomal rna (rrna) in duplex real-time pcr reactions as described. relative quantification of mrna expression was determined as the expression ratio of cytokine mrna to s rrna and compared with the same ratio measured in a calibrator sample (phytohaemagglutinin-stimulated peripheral blood mononuclear cells obtained from a healthy volunteer). induced sputum concentrations of il- and soluble icam- were measured by elisa (r&d systems, minneapolis, mn, usa), validated for the use in sputum supernatant. analysis was performed using stata (stata corporation, college station, tx, usa), with results presented as median (interquartile range) or n (%) as appropriate. significance was determined (p < . ) from chisquare test or non-parametric wilcoxon rank sum test, kruskal-wallis test or fisher's exact test. thirty-nine subjects presented with acute asthma and were assessed at recovery, a mean of . weeks later. the majority of subjects recruited for the study ( / , %), were < years of age. viral rna was detected by pcr at both visits in subjects ( . %, viral persistence) ( table ). in subjects ( . %), viral rna was detected at the acute asthma episode, but had cleared by recovery (viral clearance). subjects with viral persistence were more often male and reported less rhinitis (table ) . subjects in the viral persistence and viral clearance groups had a similar prevalence of atopy, background asthma therapy, severity of acute episode, frequency and types of viruses detected in the acute episode (tables , ) . at recovery, viral rna was detected in subjects ( . %, table ). the infectivity of the viruses was examined by their ability to infect a host cell line. at visit , . % ( / ) of the hrv isolates were able to infect a host cell line, whereas during the acute asthma exacerbation (visit ), . % ( / ) isolates were infectious (p = . ). of all the samples that were pcr positive at either visit, / ( . %) were both culture and pcr positive at visit compared with / ( . %) at visit (p = . ). these results indicate that viral nucleic acids are frequently detected at recovery for hrv infections, but not for other respiratory viral isolates, and that hrv detected at visit was less able to infect host cells than visit isolates. of the viral species detected at recovery, seven were from the same genus to the virus detected at visit ( table ) . differences in viruses isolated at the two different visits occurred due to detection of rsv (n = ) or influenza virus (n = ) at either visit, and detection of a picornavirus at the alternate visit, or detection of an enterovirus at visit and a rhinovirus at visit (n = ). where the virus detected at recovery was able to infect a host cell line, we sequenced the viral vp / regions to determine the viral serotype. in one instance the same serotype was present at visits and (hrv- ); however at visit , hrv- was detected in association with rsv type b. in another case the virus detected at recovery was a different serotype to the virus detected at the first visit: enterovirus at visit and hrv at visit . overall, eight of the subjects with viral persistence at visit had isolates that were different to the virus detected during the acute asthma exacerbation. these data indicate that when a virus is detected at recovery and is capable of infection, it is often a different virus to that associated with the acute asthma exacerbation. the clinical severity of the asthma exacerbation and the severity of the respiratory infection were similar in the viral clearance and viral persistence groups (tables , ). both groups showed clinical recovery from the asthma exacerbation and from the respiratory infection (fig. ) . tables and describe markers of airway inflammation in the subset of subjects for which adequate samples were available. the cytokine environment was altered in the viral persistence group, with elevated il- mrna and ip- gene expression ( table , fig. ). viral persistence group was not altered, with similar gene expression for il- , rantes and mip- a. similarly, protein levels of il- and icam- were similar in both groups (table ). induced sputum cell counts at recovery were similar in the viral persistence and viral clearance groups ( table ) . the impact of persistent infective virus was examined by comparing the subjects with persistent infective (culture positive) virus at recovery (visit ) to culture positive subjects at visit and culture negative subjects at visit (fig. ) . there were no differences in clinical features, sputum differentials or gene expression of ip- or il- at follow up for those culture positive compared with those negative culture (p > . ). there were also no differences in change from visit in clinical features, sputum differentials, ip- or il- gene expression for those culture positive at visit compared with those culture negative at visit (fig. ) . respiratory viral infection plays a role in > % of asthma exacerbations requiring hospitalization. the table characteristics of subjects at visit with acute asthma and viral persistence (acute asthma, virus positive at visits and ), and acute asthma with viral clearance (virus positive at visit and negative at visit ) results are median (iqr), wilcoxon rank sum test or n (%). acq, asthma control questionnaire; ccs, common cold score. main virus detected during acute exacerbation is hrv, which has been shown to be present in % of cases of virus-induced asthma. in this study, we have shown that persistence of viral nucleic acid is common in asthmatics after an acute exacerbation and is associated with an altered cytokine environment, but no persistent clinical or inflammatory abnormality. the viral rna observed most frequently to persist was hrv; however, persistence of the same infectious hrv was uncommon. when infectious virus was detected at recovery, it was often a different virus. the consequences of persistence of viral rna were examined by assessing several disease domains. the clinical severity of the exacerbation and its recovery were similar whether there was clearance or persistence of viral rna. in addition, the extent of cellular airway inflammation was similar between groups. however, viral persistence was associated with altered cytokine production, with elevated il- and ip- gene expression. these results suggest that the cellular inflammation that results from viral replication is responsible for the clinical manifestations of a viral infection in asthma, rather than viral replication itself. the period between infection by hrv and elimination of the virus with a negative pcr assay is not well described; however, hrv persistence has been seen in other circumstances. in vitro rv culture has been shown to decline rapidly (over less than days). in most experimental infections, rv shedding detected by virus culture reaches a peak around h after viral challenge, then rapidly declines, remaining positive for up to weeks. gern et al. found that experimental rv infection cleared by days in % of adults. in clinical samples, hrv rna positivity has remained detectable longer than culture positivity. among young adults with a common cold, pcr was positive after days in % of cases. enteroviruses may be shed for up to weeks from the nasopharynx and several months in faeces. asymptomatic carriage of hrv has been identified in - % of results are median (iqr), wilcoxon rank sum test. rer, relative expression ratio; see methods section for details. results are median (iqr), wilcoxon rank sum test. lg wood et al. subjects. , this could represent the early subclinical phase of a new infection, or viral persistence. in asymptomatic children with no past ( weeks) or future ( weeks) respiratory symptoms indicating a clinical infection, the viral detection rate was as low as %. persistence of rna viral infection has been previously reported. measles virus persistence has been associated with subacute sclerosing panencephalitis (sspe). , more recently persistent hepatitis c virus infection has been identified. it has been suggested that several conditions must be met for viral persistence to occur. first, the virus should infect the cell without being cytopathic. the most common persistent virus in this study was hrv. hrv typically infects a host cell without causing cell death, and in asthma, apoptosis is suppressed after hrv infection. , second, the viral genome must be maintained. for those rna viruses that do not integrate into the host genome, such as hrv, continued replication at a low level is required to maintain infection. this has been shown to occur with rsv where gene transcription continues after the acute infection. third, the virus must avoid immune detection and elimination. although specific antibodies to hrv develop after infection, their ability to protect against infection can be limited. after rsv infection, viral persistence can occur despite the presence of virus-specific igg. effective t-cell responses are critical to eradicate acute viral infections and prevent viral persistence. it has recently been shown that virus-specific cd t cells can be functionally inactivated early during the transition into viral persistence and fail to produce effector cytokines, thereby compromising an efficient and effective antiviral immune response. if these mechanisms were to occur after hrv infection, this could lead to viral persistence. when persistent viral infection does occur, there is an accumulation of viral mutations, as shown for measles, sars-cov, hepatitis c , , and enteroviral persistence. these mutations, or quasispecies, are particularly important for rna viruses as they generate high levels of genetic variation that may facilitate viral adaptation to changing environments, including the development of drug resistance or escape from the immune system. , the development of picornavirus quasispecies can favour viral persistence, as frequently these species contain deletions in the ′ntr that reduce replication to a level where cytopathic effect is not observed. , this may allow escape from the adaptive immune response that clears cytolytic wild-type virus. increasing quasispecies diversity through chemical mutagenesis was recently found to restore pathogenicity of a poliovirus isolate. it will be important in future work to characterize the rna species of hrv that persist after an asthma exacerbation and to detect viral mutations that may be associated with viral persistence. in this study the host response to viral persistence was limited. subjects with viral persistence experienced a recovery of clinical features and improvement in inflammatory cell counts, even in subjects with persistent infective virus. this observation is interesting, as it implies that there are host defence mechanisms controlling the response to virus that are adequate to inhibit viral-induced airway inflammation and clinical disease. future identification of these responses could be very important. viral persistence was associated with altered cytokine responses, with an ongoing elevation of il- and ip- gene expression. a similar observation has been made with rsv persistence, where rsv-rna could be detected in lungs of infected mice for up to days after the initial infection, associated with persistent chemokine gene transcription. the longer-term consequences of viral persistence and this modified host response warrant further study. in addition, validation of these gene expression findings with protein measurements is an important area for further research. when infectious virus was detected after recovery from acute asthma, it was often a different virus to that found during the acute exacerbation. in only one case did we confirm persistence of the same hrv serotype that was still able to infect host cells. the fact that a different virus was detected suggests that re-infection is a significant mechanism for persistent viral detection after acute asthma exacerbation. this is also supported by the observation that ip- gene expression was elevated in subjects with infective virus at visit . the role of hrv infection in leading to persistent asthma is an important and emerging research area. our data deal with adults and children aged over years studied during and post acute exacerbation. as such, we are studying populations where asthma is established and any effects of early hrv infection during the development of immunocompetence have already occurred. further research, conducted in very young children without established disease, is necessary to determine the effects of viral persistence on asthma development. in our study, % and % of subjects had hrv detected at visits and , respectively. thus the viruses identified in this study are representative of those typically associated with acute asthma. the pcr primers and probes used in this study detect all species of hrv. hence, it is possible that the three hrv species, hrv-a, hrv-b and hrv-c are represented among the samples with a generic hrv result. indeed, a recent report of virus infection in children with acute asthma found that hrv was almost universally present and identified hrv strains, including hrv-c strains. differences in the persistence of different strains and serotypes of hrv are not well understood. this highlights the need for further typing of hrv species in future work. in conclusion, we have described that rna viruses are commonly detected in airway samples after recovery from acute asthma. the viral species detected are frequently different to the infection that caused the original exacerbation; however, detection of noninfectious hrv is also common. this virus is persistent, does not elicit a host clinical response, but may alter the host cytokine environment by leading to a persistent elevation of il- and ip- gene expression. there may be longer-term effects of viral persistence in asthma. respiratory viruses and exacerbations of asthma in adults asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma pathogenesis of asthma exacerbation persistence of rhinovirus rna after asthma exacerbation in children persistence of rhinovirus and enterovirus rna after acute respiratory illnes in children use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms viral quasispecies and fitness variations quasispecies diversity determines pathogenesis through cooperative interactions in a viral population rhinovirus illnesses during infancy predict subsequent childhood wheezing interleukin- gene expression in acute virus-induced asthma development and validation of a questionnaire to measure asthma control safety of sputum induction with isotonic saline in adults with acute severe asthma isolation and identification of viruses in 'methods and techniques in virology genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus a new mathematical model for relative quantification in real-time rt-pcr clinical significance and pathogenesis of viral respiratory infections relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection the common cold: effects of intranasal fluticasone propionate treatment clinical microbiology persistence of viruses in upper respiratory tract of children with asthma molecular mechanisms of measles virus persistence identification of novel hcv subgenome replicating persistently in chronic active hepatitis c patients latency and persistence of respiratory syncytial virus despite t cell immunity intrinsic functional dysregulation of cd t cells occurs rapidly following persistent viral infection mutational escape from cd + t cell immunity: hcv evolution, from chimpanzees to man multigene tracking of quasispecies in viral persistence and clearance of hepatitis c virus ′-terminal deletions occur in coxsackievirus b during replication in murine hearts and cardiac myocyte cultures and correlate with encapsidation of negative-strand viral rna diversity, divergence, and evolution of cell-free human immunodeficiency virus type in vaginal secretions and blood of chronically infected women: associations with immune status a functional ribonucleoprotein complex forms around the ′ end of poliovirus rna weekly monitoring of children with asthma for infections and illness during common cold seasons this study was funded by the national health and medical research council of australia. key: cord- -gwtkrt u authors: mackay, ian m.; lambert, stephen b.; faux, cassandra e.; arden, katherine e.; nissen, michael d.; sloots, theo p.; nolan, terence m. title: community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a -month period date: - - journal: j infect dis doi: . /infdis/jis sha: doc_id: cord_uid: gwtkrt u human rhinovirus (hrv) replication triggers exacerbation of asthma and causes most acute respiratory illnesses (aris), which may manifest as influenza-like illness. the recent assignment of previously unknown hrv types to a third hrv species, human rhinovirus c, raised questions about the prevalence of these picornavirus types in the community, the extent of hrv diversity at a single site, and whether the hrvs have an equally diverse clinical impact on their hosts. we quantified hrv diversity, and there was no clinical impact attributable to hrv species and genotypes among a community population of preschool-aged children with ari who provided respiratory samples during . all hrv species were represented among children with ari, and distinct hrv types were cocirculating. fever accompanied . % of hrv-positive ari cases. hrvs were less likely than dna viruses to be codetected with another virus, suggesting virus interference at the community level, demonstrated by the inverse correlation between influenza virus detection and hrv detection. human rhinovirus (hrv) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease and the majority of acute respiratory illnesses (aris), some of which meet criteria for influenza-like illness. these upper and lower respiratory tract illnesses are associated with considerable direct healthcare costs and indirect costs due to time lost from and reduced performance of regular duties [ ] . there are known hrv serotypes, with - circulating simultaneously at a given site [ ] [ ] [ ] [ ] [ ] [ ] . recently, distinct, molecularly defined hrv genotypes were formally assigned to a third species, human rhinovirus c (hrv-c) [ , ] . the contribution of hrv species and individual types to the annual burden of circulating hrv is poorly defined. there are apparently distinct phylogenetic clades of hrv-c when typed using the ′ untranslated region ( ′utr) and adjoining encoding region [ ] . it is hypothesized that these clades evolved with or without genetic recombination with human rhinovirus a (hrv-a) types [ ] . nevertheless, each available majority sequence of an hrv-c type has to date represented a genetically unique, phylogenetically distinct, and globally distributed virus detected in patients with aris. there are specific seasonal and annual variations in respiratory virus circulation and interactions [ ] [ ] [ ] . in a retrospective pediatric hospital-based study, hrvs were found to be statistically least likely of examined viruses to be codetected with another virus during [ ] . this aspect of virus-to-virus interaction is described as virus interference and is hypothesized to be the result of the host's response to one virus diminishing the likelihood of infection by another virus [ , ] . for hrvs, virus-to-virus interaction was once exploited as an in vitro diagnostic tool, whereby successful experimental hrv infection of organ cultures was indicated by blockading the replication of another, superinfecting respiratory virus [ ] . we sought to quantify the genetic diversity, epidemiology, and impact of hrv and enterovirus species, conjointly referred to hereafter as picornaviruses, circulating among a community cohort of preschool-aged children who provided respiratory samples over a -year period. we also sought to build on our hospital-based virus-to-virus interaction analyses [ ] by seeking preliminary observational evidence of virus interference within the community. after receipt of informed consent from the parent or guardian of a potential study subject, we enrolled healthy children < years of age into a community-based dynamic cohort study conducted over months in melbourne, australia [ ] . the study commenced in january , and enrollment was progressive and continued until november , with children observed until january . parents monitored a set of symptoms in the study child each day, and when the definition of ari was met we asked parents to collect a combined nosethroat swab specimen [ ] . the specimen was couriered to the victorian infectious diseases reference laboratory, where it underwent conventional polymerase chain reaction (pcr) testing, with reverse-transcription pcr performed for rna viruses, including influenza a and b viruses, respiratory syncytial virus, parainfluenza viruses, hrvs, and enteroviruses [ ] . pcr was conducted for adenoviruses [ ] . illnesses were classified as aris uncomplicated or complicated by fever and/ or otitis media. at the completion of data collection, we transported all available specimens (original nose-throat swab specimens and complementary dna) on dry ice to the queensland paediatric infectious diseases (qpid) laboratory, where they were tested for metapneumovirus and coronavirus nl (hereafter, "coronavirus"), using real-time pcr [ ] . hrv templates for pcr amplification at the qpid laboratory were complementary dna, created at the victorian infectious diseases reference laboratory during the original studies [ ] , or fresh rna extracted by means of the corbett x-tractor gene system (corbett research, australia) from the original nose-throat swab specimen. all extracts were amplified using a broadly reactive screening assay that targets the ′utr [ ] , yielding amplicons (length, approximately base pairs) for sequencing by the prism bigdye sequencing kit v . (applied biosystems). sequences generated by this study and submitted to genbank included accession numbers jn - and jq - . sequence analysis was conducted in geneious pro [ ] . the picornavirus species was determined by best match, using the following algorithm: when a basic local alignment search tool (blast) comparison returned matches with ≥ % sequence identity in the ′utr, with characterized members assigned to a given species, our sequence was assigned to that species. for viruses not classified at this stage, we used definitive ′utr-derived data from lee et al [ ] as a guide to classify any query sequence that shared > % nucleotide identity in the ′utr with a blast match as a variant of that hrv type. counts and proportions were recorded for descriptive analyses. similar to methods we described previously [ ] , univariate analysis involving the χ test or the fisher exact test, using × contingency tables, was used to evaluate the relationships between picornavirus type, season, and demographic variables, such as age and sex, as well as to study virus codetection with another virus. a p value of <. was considered to indicate a statistically significant association. we observed child-days in just over study months. there were aris identified; in cases ( %), at least parent-collected combined nose-throat swab specimen was returned [ ] . for aris ( specimens; . % of specimens), hrv or enterovirus was identified by conventional pcr at victorian infectious diseases reference laboratory [ ] . of specimens previously positive for a picornavirus and shipped to qpid laboratory, ( . %) yielded a genotypable sequence (figure ), ( . %) yielded uninterpretable sequences, and ( . %) yielded sequences that were not amplifiable [ ] . the picornavirus-positive specimens originated from children (mean age, . months), including from infants (age, - months), from toddlers (age, - months), and from older children (age, - years). a mean of . picornaviruses (range, - ) were detected per child. sixty picornavirus-positive children ( . %) had ≥ picornaviruses detected during study participation, and had ≥ (table and figure ). no child was positive for the same hrv type during different ari episodes, although the same species might have been detected. of the genotyped viruses, ( . %) were hrv-a, ( . %) were human rhinovirus b (hrv-b), ( . %) were hrv-c, and ( . %) were enteroviruses ( figure ). picornavirus species distributions were not associated with sex (table ). there was a decreased likelihood of identifying any of the picornavirus species in infants and an increased risk of identifying hrv-b, hrv-c, and enteroviruses in older children (p < . for these associations). the proportion of ari episodes in which hrv-b and hrv-c were detected was lowest during summer, while the proportion of ari episodes in which hrv-a was detected was lowest in both summer and winter (figure ). detection of each hrv species declined dramatically when detection of respiratory syncytial virus and metapneumovirus increased and detection of influenza a virus peaked (in august [ ] ). hrv-a and hrv-c could be detected in subsequent ari specimens from the same child, but hrv-b positivity did not recur for any child. peak picornavirus activity occurred in may and october, approximately weeks after school terms commenced. activity of the species were observed to peak distinctly (species exchange) during the course of the year. hrv-positive ari cases were accompanied by fever in instances ( . % of all aris; . % of hrv detections; table ), whereas the majority of cases ( [ . %] of ) in which enterovirus types (including coxsackievirus a , a , a , and b ; echovirus , , , , and ; and poliovirus ) were identified had fever reported. only cases of wheeze were reported: were associated with hrv-c, was associated with enterovirus, and were associated with untypable picornaviruses. otitis media without fever accompanied ari in picornavirus-positive instances ( . %), and fever and otitis media co-occurred with ari in instances ( . %). hrv-a, hrv-b, and hrv-c were detected in similar proportions of ari cases with complications ( . %, . %, and . % respectively) and those without complications ( . %, . %, and . % respectively). of samples with any virus present, ( . %) included a second virus, whereas ( . %) were positive for viruses. among picornavirus-positive specimens, ( . %) were pcr positive for another virus; ( . %) were not detected with another virus. only respiratory syncytial virus was codetected with another virus on fewer occasions ( . % of respiratory syncytial virus detections). despite this, among children with ≥ hrv detection during enrollment ( children had pvs; had pvs; had pvs, had pvs and had pvs), there was a consistent pattern of a reduced likelihood of codetection with a number of other viruses. for example, an hrv detection was associated with a reduced likelihood of codetecting any of viruses or virus groups (adenovirus, parainfluenza virus, respiratory syncytial virus, influenza a virus, metapneumovirus, or coronavirus), as reflected by an odds ratio of < (table ). among picornaviruses, enterovirus was most frequently associated with codetection ( % of enterovirus detections), and hrv-b was least frequently associated with codetection ( % of hrv-b detections). there was an average of days between consecutive aris in study children. after each hrv, influenza a virus, respiratory syncytial virus, or adenovirus detection, there was a mean interval of , , , and days, respectively, before the next ari episode. the evolutionary history was inferred using the neighbor-joining method in mega [ , ] . the optimal tree is shown drawn to scale, with branch lengths in the same units as those of the evolutionary distances (base substitutions per site; maximum composite likelihood method [ ] ) the analysis involved nucleotide sequences, including completely sequenced referenced types [ ] . our analysis of aris among children in the community identified named or uncharacterized rhinovirus types among children followed during this -month study. three-tofive times more distinct hrv types were identified in our single-year cohort than would have been expected from the literature. recently, van der zalm et al identified different subtypes among a cohort of children sampled fortnightly during a six-month period [ ] . because a lower sequence threshold was used to identify those viruses, it is likely that the true number of hrv types was underestimated as compared to the higher threshold ( % vs %) we applied, which we considered essential when using the highly conserved ′ utr region. recent cohort studies that also used the ′utr for hrv typing identified [ ] distinct hrv types, including new types [ ] , whereas other cohort studies have not conducted detailed typing analyses [ , ] . cohorts with longer follow-up [ ] or more-frequent sampling than ours [ ] unsurprisingly find greater numbers of hrv types since they span multiple seasons or have a better likelihood of sampling so-called "asymptomatic" infections. frequently, sampling in longitudinal cohort studies improves our understanding of the role of respiratory viruses in illness because it allows temporal association with signs and symptoms. despite the dominance of hrv-c types here and their reported links with asthma elsewhere [ ] , few new cases of wheezing were reported in our cohort, most likely because of exclusion of children with chronic pulmonary disorders (including diagnosed asthma or frequent use of asthma medication). a frequently sampled, well-controlled longitudinal cohort of children with asthma will be required to answer whether there is a species-specific hrv impact in an asthmatic population. all hrv species were represented among ari cases with fever, providing further evidence of the confounding capacity of hrv infections during an influenza epidemic or pandemic unless specific diagnostics are included. most respiratory viruses were detected in toddlers and children, from whom the majority of specimens were obtained. in our study, the respiratory syncytial virus and metapneumovirus ari with fever and om ( . season preceded the influenza a virus season. it was also noteworthy that hrv prevalence declined sharply as respiratory syncytial virus, metapneumovirus, and, particularly, influenza a virus cases peaked. this mirrored the findings from our hospital studies, conducted on specimens from brisbane, queensland, australia, that were also collected during [ ] . we observed that viruses with an rna genome, in particular the hrvs, were less frequently involved in codetections than those with a dna genome in this community cohort from victoria. these numbers might have been different if other virus, such as bocavirus, parainfluenza virus- , and influenza c virus, had been included, but our previous findings suggest the adjustment would not have changed the significance of the virus-to-virus interaction [ ] . our observation most likely reflects the capacity of rna viruses to efficiently trigger the early innate interferon response through interactions with a range of pattern-recognition receptors, including toll-like receptors and , mda , and rig-i. whether dna viruses are better adapted to interfere with a host interferon response or are better at exploiting the opportunities provided by rna viruses remains to be defined. it has been previously suggested that the ′utr is unsuitable for hrv genotyping because in silico signs suggest that the region has, at some point, been a site for recombination between some hrv types [ ] . however, we have not found, nor has there been published evidence for, false genotyping (a ′utr erroneously representing a polyprotein sequence from a different virus) of the ≥ hrv-a, hrv-b, and hrv-c genomes fully sequenced to date. even when an hrv-c type falls into the hrv-a clade phylogenetically, to date it exists within a distinct space in that clade. carefully chosen sequence identity thresholds and experience can successfully produce robust genotyping results, as has been shown here and elsewhere in the literature [ , , , [ ] [ ] [ ] . in this study, we found that each ′utr sequence was a unique identifier of hrv type. others suggest the hrvs are under positive selective pressure to remain conserved [ ] . while this may not remain evident as more completed hrv genomes become available, no proof exists to support a theory that the ′utr is an unsatisfactory target for genotyping, and it continues to be used successfully elsewhere [ ] . nevertheless, phylogeny based on the ′utr region, whether partial or complete, does not accurately discriminate all members of the hrv-a species from those currently considered to be hrv-c but harboring elements of an hrv-a utr [ ] . this is similar to the phylogenetic patterns of enterovirus types derived from use of the ′utr, in which the known species are represented by only clades. a longer target encompassing a ′utr-vp stretch improves phylogenetic tree construction, and these sequences are becoming well represented in the genbank sequence database [ ] . however, until the hrv virome is completely characterized, it is also premature to assume that the ′utr-vp region's apparent congruity with the rest of an hrv's polyprotein sequence will be maintained by the genomes of future distinct hrv strains. a vp target is less sensitive when used directly on clinical specimens [ ] and is likely to underrepresent enterovirus diversity [ ] . in summary, our screening and ′utr-based genotyping study identified extensive hrv genetic diversity as compared to data generated from culture-based diagnostics. there were no associations between sex or clinical outcome and hrv species, but hrvs were less often present in codetections than would have been expected by chance. in particular, the influenza a virus season preceded a precipitous decline in hrv detections. whether a reduction in hrvs allows an increase in other viruses during the winter months is unknown, and studies to define the immunobiological mechanisms behind these observations are required. the cost of communitymanaged viral respiratory illnesses in a cohort of healthy preschool-aged children rhinovirus infections in an industrial population. . number and prevalence of serotypes screening and comprehensive vp / -typing of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp -vp typing reveals high incidence and genetic diversity hrv species c virologic studies of acute respiratory disease in young adults. iv virus isolations during four years of surveillance rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study clinical features and complete genome characterization of a distinct human rhinovirus genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) newly identified human rhinoviruses: molecular methods heat up the cold viruses interference between outbreaks of respiratory syncytial virus and influenza virus infection interference between outbreaks of epidemic viruses influenza in children relationship to other respiratory agents do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? interference between animal viruses a systematic approach to virus-virus interactions rhinoviruses and common colds parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research community epidemiology of human metapneumovirus, human coronavirus nl , and other respiratory viruses in healthy preschool-aged children using parentcollected specimens amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illness in infants association between human rhinovirus c and severity of acute asthma in children risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study epidemiology of documented viral respiratory infections and acute otitis media in a cohort of children followed from two to twenty-four months of age prevalence of and risk factors for human rhinovirus infection in health aboriginal and non-aboriginal western australian children serial viral infections in infants with recurrent respiratory illnesses a novel group of rhinoviruses is associated with asthma hospitalizations evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection assay for ′ noncoding region analysis of all human rhinovirus prototype strains increased h n infection rate in children with asthma molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c genome-wide diversity and selective pressure in the human rhinovirus lower airway rhinovirus burden and the seasonal risk of asthma exacerbation proposals for the classification of human rhinovirus species c into genotypically-assigned types new respiratory enterovirus and recombinant rhinoviruses among circulating strains mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods the neighbor-joining method: a new method for reconstructing phylogenetic trees prospects for inferring very large phylogenies by using the neighbor-joining method sequencing and analyses of all known human rhinovirus genomes reveals structure and evolution acknowledgments. we thank the families and children who participated in this study; kelly allen, other virgo staff, maternal key: cord- -sras r z authors: saraya, takeshi; kimura, hirokazu; kurai, daisuke; ishii, haruyuki; takizawa, hajime title: the molecular epidemiology of respiratory viruses associated with asthma attacks: a single-center observational study in japan date: - - journal: medicine (baltimore) doi: . /md. sha: doc_id: cord_uid: sras r z few reports have described the significance of viral respiratory infections (vris) in exacerbation of asthma in adult patients. the aim of this study was to elucidate the profiles of vris in adult patients with asthma along with their molecular epidemiology. a cross-sectional observational study was conducted at kyorin university hospital from august to may . to identify respiratory pathogens in inpatients and outpatients suffering from asthma attacks, rt-pcr/sequencing/phylogenetic analysis methods were applied alongside conventional microbiological methods. phylogenetic and pairwise distance analyses of viruses were performed. a total of asthma attack patients enrolled in this study in both inpatient (n = ) and outpatient (n = ) settings. the total respiratory samples were obtained from nasopharyngeal swab (n = ) or sputum (n = ). among these, patients with virus alone (n = ), virus and bacterial (n = ), and bacterial alone (n = ) were identified. the ratio of virus-positive patients in inpatient or outpatient to the total cases were . % (n = ) and . % (n = ), respectively. the frequency of virus-positive patients was significantly higher in inpatients ( . %, n = ) than in outpatients ( . %, n = ). major vris included human rhinovirus (hrv) (n = ), human metapneumovirus (hmpv) (n = ), influenza virus (inf-v) (n = ), and respiratory syncytial virus (rsv) (n = ) infections with seasonal variations. hrv-a and hrv-c were the most commonly detected viruses, with wide genetic divergence on phylogenetic analysis. asthmatic exacerbations in adults are highly associated with vris such as hrv-a or hrv-c, hmpv, rsv, and inf-v infections with seasonal variations and genetic divergence, but similar frequencies of vris occurred in asthma attack patients throughout the seasons. risk factors relating to the development of asthma attacks are multiple and complex. the frequency of viral respiratory infections (vris) in asthma attack patients is now recognized as having a major impact on asthma pathogenesis in children. [ ] kusel et al [ ] reported that vris caused by human rhinovirus (hrv) and respiratory syncytial virus (rsv) in the first year of life were strongly associated with the diagnosis of current asthma and persistent wheeze in -year-old children. furthermore, jackson et al [ ] demonstrated that children had an increased risk of asthma at years of age if they experience wheezing in the first years of life, with rsv [odds ratio (or): . ], hrv (or: . ), or both hrv and rsv (or: ). regarding adult asthma patients, previous reports showed that respiratory tract infections associated with asthma exacerbation ranged from % to % to %, [ ] [ ] [ ] of which % are hrv. [ ] however, especially in japan, few epidemiological data exist on the correlation between vris and asthma attacks in adult patients [ , ] from studies conducted within the year. [ ] moreover, martin et al [ ] and kraft et al [ ] described the correlation between mycoplasma pneumoniae infection and asthma attacks in adult patients; however, the preliminary findings could not confirm the evidence of m. pneumoniae infection in those patients. [ , ] therefore, we prospectively studied adult patients with asthma attacks to clarify the role of vris and/or bacterial infection, including m. pneumonia, and their molecular epidemiology. in this cross-sectional observational study, we prospectively enrolled adult patients suffering from asthma attacks visiting kyorin university hospital (a -bed tertiary center in tokyo) in both inpatient and outpatient settings from august to may . eligible patients were aged over years and had a clinical diagnosis of asthma in addition to one or more of the following characteristics: variability in peak expiratory flow of more than %; airway reversibility by inhaled b agonist; hyperresponsiveness to methacholine challenge; and recurrent dyspnea episodes with wheezing. asthma attack patients were enrolled in this study if they had acute or subacute episodes of progressively worsening shortness of breath, cough, wheezing, and chest tightness, or some combination of these symptoms, characterized by decreases in expiratory air-flow and objective measures of lung functions according to the latest national institute of health (nih) national asthma education and prevention (naep) guidelines/global initiative for asthma (gina) guideline or satisfied the moderate to severe exacerbation of asthma based on the american thoracic society (ats)/european respiratory society (ers) statement. [ ] we excluded the patients who had chronic obstructive lung disease, pneumonia, interstitial lung diseases, acute heart failure, and respiratory symptoms that were possibly due to infections in the last month. respiratory samples included sputum or nasopharyngeal swab collected at inpatient admission or at the outpatient setting at the time patients were diagnosed of asthma attacks. clinical data were also obtained at the same time. respiratory samples for pcr-based detection of respiratory viruses, m. pneumoniae, and chlamydophila pneumoniae were collected separately from those intended for bacterial cultures and were stored at - °c until use. gram stain was performed on a purulent portion of each sputum specimen and examined by trained personnel. sputum samples were considered as good quality for evaluation if they were classified as geckler or . positive bacterial culture was based on acceptable sputum samples with predominant species and compatible results from gram staining. samples were centrifuged at g at °c for minutes. viral rna and dna were extracted from supernatants using the qiaamp viral rna mini kit (qiagen, valencia, ca). reverse transcription was performed using primescript rt reagent kit (takara bio, otsu, japan), according to the manufacturer's instructions. we used reverse transcription polymerase chain reaction [(rt)-pcr] to try to detect both dna and rna viruses, including human metapneumovirus (hmpv), human rhinovirus (hrv), enterovirus, respiratory syncytial virus (rsv), influenza viruses a, b, and c (inf-a, b, and c), human parainfluenza viruses, human coronavirus, adenovirus, cytomegalovirus, human parvovirus b , varicella zoster virus, and human bocavirus together with m. pneumoniae and c. pneumoniae as previously described. [ , ] we used specific primer sets for the amplifications of these viruses as we also previously described. [ , ] pcr products were purified using monofas dna purification kit i (gl sciences inc., shinjuku, tokyo, japan). the purified products were sequenced with a bigdye terminator v . cycle sequencing kit (applied biosystems, foster city, ca) using the above primers. sequence analysis was performed on an abi genetic analyzer (applied biosystems). the nucleotide sequences thus obtained were given genbank accession numbers from lc to lc . . . the species of the viruses were estimated using the basic local alignment search tool (blast). [ ] evolutionary distances were estimated using the kimura -parameter method and phylogenetic trees were constructed using the neighbor-joining (nj) method. reliability of the trees was estimated using , bootstrap replications. we calculated pairwise distances (p-distance) of the respiratory viruses detected in this study, hmpv, rsv, inf-v, and hrv, using mega software, version . . calculations were based on the nucleotide sequences of each virus. samples were collected after written informed consent was obtained from the subjects or their legal representatives. the study protocol was approved by the ethics committee on human research of kyorin university hospital (h - ) on july , . the protocols were carried out in accordance with the approved guidelines. statistical comparisons of nonparametric data were performed using the mann-whitney test or wilcoxon signed-rank test. comparisons of categorical data were made using pearson chi-squared test. all tests were -sided. a value of p < . was considered statistically significant. data were analyzed using ibm spss version . software for windows (spss, chicago, il). we examined a total of asthma attack patients in the study period, in both inpatient (n = ) and outpatient (n = ) settings. among these, patients with virus alone (n = ), virus and bacterial (n = ), and bacterial alone (n = ) were identified. the ratio of virus-positive patients in inpatient or outpatient to the total cases were . % (n = ) and . % (n = ), respectively. the frequency of virus-positive patients was significantly higher in the inpatient group ( . %, n = ) than in the outpatient group ( . %, n = ) ( table ) . patient age, sex, and the proportion of smokers were similar in both groups. however, the proportion of patients with hypoxemia (spo %), wheezes, and patients classified as severe or serious asthma attack according to japanese guideline [ ] and/or ats/ers statement [ ] were significantly higher in the inpatient group than in the outpatient group ( count and c-reactive protein (crp), were also comparable in both groups. of the inpatients, ( . %) were virus positive ( table ) . the age and male to female ratio was comparable between viruspositive and virus-negative inpatients. duration of asthma in years (median , iqr - vs median , iqr . - . , p = . ) and the proportion of smokers ( . % vs . %, p = . ) were not significantly different in virus-positive and negative inpatients. in addition, the proportion of the patients with hypercapnia (paco ≥ torr) was not significantly higher in virus-positive inpatients than in virus-negative inpatients ( . % vs . %, p = . ). duration of respiratory failure (days), wheezes (days), steroid treatment (days), and hospital stays (days) did not differ significantly in virus-positive and negative inpatients (table ) . furthermore, the value of serum inflammatory markers on admission, such as wbc count and crp, were equal in both groups. the total respiratory samples were obtained from the nasopharyngeal swab (n = ) or sputum (n = ). among the sputum samples, good quality samples (geckler classification of or ) were only samples, out of which samples were positive for bacteria (table ) . within the study period, we identified a total of virus detection events obtained from the inpatient or outpatient settings. the virus detection event consisted of hrv (n = ), hmpv (n = ), inf-v (n = ), and rsv (n = ) ( table ). the main hrv species were identified as hrv-a (n = ) and hrv-c (n = ), with only hrv-b infection. among the virus-positive group (n = ), the incidence of bacterial coinfection (bacteria table comparison of the clinical characteristics of inpatient and outpatient asthma attack patients. inpatients (n = ) outpatients (n = ) p during the study period (approximately years), respiratory virus infection was more common in the spring (march to may) and autumn (september to november) (fig. ) . of note, the frequency of hrv and rsv infection seemed to be high in autumn, while hmpv was likely to be indicated in spring. similarly, inf-v infection was most common in the winter and spring. the proportion of virus-positive patients to the total asthma attack subjects in each season was . % (n = , spring), % (n = , summer), . % (n = , autumn), and . % (n = , winter). however, the incidence of virus infection in each season was not statistically significant. we performed phylogenetic analysis of hrv, hmpv, inf-v, and rsv as a major cause of asthma attack in this study. hrv was the most commonly detected virus and consisted of diverse genotypes on phylogenetic analysis (fig. ) . this trend was also found in the analyses of hmpv (fig. ) , inf-v (fig. ) , and rsv (fig. ). this study was the first to show that various respiratory viruses such as hrv, hmpv, inf-v, and rsv were associated with asthma attacks in japanese adult patients. hrv was the most common cause of asthma attacks in autumn, inf-v was predominantly seen in winter to spring, and vris seemed to equally affect asthma attacks in every season in japan during the -year study period. moreover, the molecular epidemiological data suggested that the detected viruses had a wide genetic divergence (particularly hrv-a and hrv-c) with seasonal variations. fujitsuka et al [ ] showed that a majority of japanese children with acute wheezing illness during a -month period had an rsv, hrv, or rsv and hrv infections. they also found that rsv was dominantly detected in patients with no history of wheezing and/or asthma, while hrv was dominant in patients with a history of wheezing and/or asthma. indeed, the present study demonstrates that hrv is an important cause of asthma attacks, even in adult patients. the molecular epidemiology of each hrv species associated with asthma attacks may not be completely known, because the hrv-c was recently recovered or nonculturable. [ et al [ ] found that hrv-c has a stronger association with virusinduced asthma than hrv-a and hrv-b in hospitalized children younger than years with acute respiratory illness. however, the epidemiological data of hrv associated asthma attacks in adults were scarcely reported in japan. in this regard, the present study showed that hrv-a or hrv-c has a potential role in asthma attacks among japanese adult patients, with a wide genetic divergence. as shown in fig. , the detection of hrv was most common in autumn, indicating that the common cold, which results from hrv during the fall months, can lead to asthma attacks as reported in previous studies. [ ] furthermore, previous reports described other respiratory viruses, such as hmpv, [ ] rsv, parainfluenza virus, inf-v, and coronavirus, as a cause of asthma attack in adults, [ ] and the present study clearly demonstrated the main causative viruses are hrv, hmpv, rsv, and inf-v. what is more important is the finding in our study that the various vris can cause asthma attacks with a seasonal predilection in each virus; however, the frequency of vris in asthma attack patients seem to be equal throughout the seasons. interestingly, there was no infection or coinfection with m. pneumoniae or c. pneumoniae, which is contrary to the findings described in previous reports. [ , ] of note, regarding inpatients, virus infection seemed to be associated with hypercapnia (paco ≥ torr) and the duration of asthma (years), but the severity of wheezing and/or bronchial epithelial damage differed among respiratory viruses, [ ] thereby furthering the accumulation of index cases required to independently demonstrate the pathogenesis of asthma attacks or the clinical characteristics of each virus. the limitations of our study include lack of data for the severity of bronchoconstriction or inflammation in the lower respiratory tract and paucity of data about the different mechanisms of asthma attacks resulting from each virus infections, which might lead to the diverse respiratory symptoms or the development of airway remodeling. however, the present study reports the first evidence of vris in asthmatic adult japanese patients with a clear molecular epidemiology in a single-center cohort. asthmatic exacerbations in adults are highly associated with vris, such as hrv-a or hrv-c, hmpv, rsv, and inf-v infections with seasonal variations and genetic divergence, but the frequency of vris in asthma attacks seemed to be equal throughout the seasons. virus-induced exacerbations in asthma and copd early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma wheezing rhinovirus illnesses in early life predict asthma development in high-risk children respiratory tract viral infections in inner-city asthmatic adults respiratory viruses and exacerbations of asthma in adults the incidence of respiratory tract infection in adults requiring hospitalization for asthma viruses in asthma exacerbations epidemiology of virus-induced asthma exacerbations: with special reference to the role of human rhinovirus the importance of bacterial and viral infections associated with adult asthma exacerbations in clinical practice a link between chronic asthma and chronic infection detection of mycoplasma pneumoniae in the airways of adults with chronic asthma is mycoplasma pneumoniae infection associated with adult asthma exacerbation? pathogen profiles and molecular epidemiology of respiratory viruses in japanese inpatients with community-acquired pneumonia a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness japanese guideline for adult asthma an official american thoracic society/european respiratory society statement: asthma control and exacerbations: standardizing endpoints for clinical asthma trials and clinical practice clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children a novel group of rhinoviruses is associated with asthma hospitalizations frequency and natural history of rhinovirus infections in adults during autumn human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults key: cord- -v t b ca authors: benkouiten, samir; charrel, rémi; belhouchat, khadidja; drali, tassadit; salez, nicolas; nougairede, antoine; zandotti, christine; memish, ziad a.; al masri, malak; gaillard, catherine; parola, philippe; brouqui, philippe; gautret, philippe title: circulation of respiratory viruses among pilgrims during the hajj pilgrimage date: - - journal: clin infect dis doi: . /cid/cit sha: doc_id: cord_uid: v t b ca background. the hajj is the oldest and largest annual mass gathering in the world and may increase the risk of spread of respiratory viruses. methods. we performed a prospective survey among a cohort of pilgrims departing from marseille, france, to mecca in the kingdom of saudi arabia (ksa) for the hajj season. nasal swabs were collected from participants and tested for respiratory viruses by real-time reverse transcription polymerase chain reaction. results. of participants sampled before departing to the ksa, ( . %) were positive for at least virus ( rhinovirus, influenza c, adenovirus, and enterovirus). seventy symptomatic pilgrims underwent additional nasal swabs during their pilgrimage in the ksa, of which ( . %) were positive for at least virus ( rhinovirus, influenza a, influenza c, respiratory syncytial virus b, metapneumovirus, adenovirus, and enterovirus). this was significantly higher than the . % who were positive before departing for the ksa (p < . ). of pilgrims sampled before leaving the ksa, ( %) were positive for at least virus ( rhinovirus, adenovirus, influenza b, and enterovirus), which was also significantly higher than the percentage of positive pilgrims ( . %), before departing for the ksa (p = . ). conclusions. this study suggests a rapid acquisition of respiratory viruses among pilgrims during their stay in the ksa, most notably rhinovirus, and highlights the potential of spreading these infections in the pilgrims' home countries upon their return. the hajj and its rituals are physically demanding and usually last for week, but the period of pilgrimage is up to month. on arrival in mecca, the holiest city in islam, hajj pilgrims start their pilgrimage by visiting the grand mosque for the circumambulation (tawaf ) of the kaaba. the pilgrims then move, during subsequent days, to different sacred places located around the city of mecca, including mina, arafat, and the muzdalifah valleys, where they reside for several nights in tent camps [ ] (figure ). later, most pilgrims leave mecca for the city of medina to visit islam's secondholiest site, the mosque of the prophet. the hajj presents major public health and infection control challenges. in addition to fatigue and extreme weather conditions [ ] (in mecca, during october the average temperature is > °c during the day and > °c at night, with a monthly rainfall averaging mm), which increase the susceptibility of pilgrims to airborne infections, inevitable overcrowding within a confined area of individuals from different parts of the world and close contact with others greatly increase the risk of acquiring or spreading infectious diseases during the pilgrims' stay [ ] ; in particular, acute respiratory infections, which are among the leading cause of acute illnesses worldwide, can spread among the pilgrims. respiratory tract infections are very common during the hajj [ , ] and account for most of the hospital admissions during this period [ , ] . it has been estimated that more than one-third of pilgrims will experience respiratory symptoms during their stay [ ] . a range of pathogens can cause acute respiratory infections, but respiratory viruses were found to be the most common etiology of upper respiratory tract infections among pilgrims in several surveys [ ] . this study, including sample collection and laboratory methods, was conducted among a cohort of pilgrims departing from marseille, france, to mecca in the ksa for the hajj season. they were tested for the most common respiratory viruses, with the aim of elucidating the dynamics of viral circulation among pilgrims. participants were recruited between august and october from a private specialized travel agency in the city of marseille, france, which organizes travel to mecca. pilgrims who planned to take part in the hajj season were asked to participate in the study on a voluntary basis if they were years of age or older and were able to provide consent. we conducted a cohort survey of participants who were followed up and sampled before departing to the ksa, during their pilgrimage in the ksa, and just before leaving the ksa. upon inclusion, the participants were questioned by arabicspeaking investigators using a standardized pretravel questionnaire, which included demographic data and medical history. a posttravel questionnaire, which collected travel-associated diseases, vaccination status, and compliance with protective behaviors, was completed by a french muslim arabic-speaking medical doctor who traveled with the pilgrims. this survey was administered during a face-to-face interview just prior to returning to france or via telephone after returning to france. if we were unable to contact the pilgrim after attempts, we considered the pilgrim lost to follow-up. health problems that occurred during the stay in the ksa were monitored and recorded by the medical doctor. a cough was defined as the occurrence of a cough with or without sputum. subjective fever was defined as a pilgrim's report of feeling feverish. for the purpose of this study, influenza-like illness (ili) was defined according to the presence of the triad of a cough, sore throat, and subjective fever [ ] . this study was approved by our local ethics committee under number - and by the saudi ethics committee. it was performed in accordance with the good clinical practices recommended by the declaration of helsinki and its amendments. all participants gave written informed consent. anterior nare swabs were systematically collected from each participant using a commercial rigid cotton-tipped swab applicator (sigma virocult, mw s, wiltshire, england), in the month before departing from france and in the days before leaving the ksa. in a number of cases, the medical doctor also collected additional nasal samples at the onset of symptoms during the pilgrimage in the ksa (within weeks after their arrival). all the samples collected during the study were placed in viral transport media (virocult virus transport medium) at the point of collection and kept at room temperature (stabilized by air conditioning to °c, in france and in the ksa) before being transported to the marseille laboratory for storage in a − °c freezer. each sample was tested for the following viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr): influenza a (flua) [ ] , influenza b (flub) [ ] , influenza c (fluc), and a/ /h n [ ] viruses; human respiratory syncytial virus a and b (rsvb) [ ] ; human metapneumovirus (hmpv) [ ] ; human rhinovirus (hrv) [ ] ; ms bacteriophage; human adenovirus (hadv) [ ] ; and human enterovirus (hev) [ ] . total nucleic acids were purified from a -μl sample volume and were spiked with ms + t bacteriophage as an internal control [ ] , using the biorobot ez xl with the virus mini kit v . (both from qiagen, courtaboeuf, france) according to the manufacturer's instructions. each sample was tested independently in a -μl reaction containing μl of rna, . μl of × buffer (iscripttm one-step rt-pcr kit for probes, bio-rad), μl of reverse transcriptase/taq, nm concentration of each primer, and nm of probe. the reactions were performed using a c tm thermal cycler (cfx tm real-time system, bio-rad, marnes-la-coquette, france). the following cycling conditions were applied: °c for minutes, followed by °c for minutes; and then cycles of °c for seconds and °c for seconds. the presence of inhibitors was determined using ms and t bacteriophage-specific detection systems, as previously reported [ ] . we hypothesized that several factors may influence the outcome of respiratory symptoms or virus portage during the stay, including age, preventive measures, and underlying chronic diseases. the pearson χ test and fisher exact test, as appropriate, were applied to analyze the categorical variables. p values of ≤. were considered significant. statistical analyses were performed using spss software, version . . a total of participants were recruited to take part in the study, of whom responded to the pretravel questionnaire. table summarizes their baseline demographics and characteristics. participants' mean age was . years (sd, . ; range, - years), with a male-to-female sex ratio of . to . they were predominately born in north africa ( . %), and most of the foreign-born individuals reported living in france for > years. most of the participants reported living in marseille and the surrounding cities. more than half of the participants ( . %) reported suffering from at least chronic disease, including diabetes ( . %), hypertension ( . %), chronic respiratory disease ( . %), and chronic cardiac disease ( . %). a total of posttravel questionnaires were completed, representing a total response rate of . %, and . % of these were telephone-administered. the mean time between return from the ksa and administration of the questionnaire by telephone was days (range, - days). before departing to the ksa, none of the pilgrims presented acute respiratory symptoms at the time they were sampled. pilgrims stayed in the ksa for weeks, and the vast majority ( . %) of them suffered from at least respiratory symptom during their stay. a cough was the most frequently reported complaint ( . % of respondents), followed by sore throat ( . %), rhinorrhea ( . %), myalgia ( . %), feverishness ( . %), dyspnea ( . %), conjunctivitis ( . %), and diarrhea ( . %). of the pilgrims who reported respiratory symptoms during their stay in the ksa, . % met the criteria for selfreported ili. the onset of respiratory symptoms peaked at - days after the arrival of the pilgrims in mecca and declined thereafter. a second peak of smaller amplitude occurred on days and after arrival in mina, shortly after moving from arafat and muzdalifah. both peaks immediately occurred after performing the tawaf in mecca. finally, only % of the pilgrims who were systematically sampled before leaving the ksa reported respiratory symptoms during the days prior to leaving the ksa. regarding preventive measures, . % of participants reported receiving a seasonal influenza vaccination in the past year and . % reported receiving a pneumococcal vaccination in the past years. during their stay in the ksa, . % of pilgrims reported using facemasks, . % using disposable handkerchiefs, . % frequent hand-washing, and . % using hand sanitizer. pilgrims who reported frequent hand-washing during their stay, compared with those who reported using typical handwashing habits, more frequently reported feverishness ( . % vs . %; odds ratio [or], . ; % confidence interval [ci], . - . ; p = . ) and ili symptoms ( . % vs . %; or, . ; % ci, . - . ; p = . ). rhinorrhea was more frequently reported by pilgrims who declared that they were not vaccinated against influenza in compared to vaccinated pilgrims ( . % vs . %; or, . ; % ci, . - . ; p = . ). dyspnea was more frequently reported by those with chronic respiratory disease compared to other pilgrims ( . % vs . %; or, . ; % ci, . - . ; p = . ). the majority of pilgrims ( . %) sought healthcare from a doctor during their stay in the ksa, and . % consulted a doctor after their return to france. four pilgrims ( %) were hospitalized ( in the ksa and upon returning to france), for respiratory tract infection, for ili symptoms, for nephritic colic, and for vomiting. no deaths occurred. of the participants enrolled in the study, ( . %) underwent a systematic pre-hajj nasal swab before traveling to the ksa, and ( . %) underwent a systematic post-hajj nasal swab in the days before leaving the ksa ( . % underwent both pre-and post-hajj nasal swabs). a total of pilgrims ( . %) also underwent an additional nasal swab at the onset of acute respiratory symptoms during their pilgrimage in the ksa. the collection dates of the samples and results are shown in figures , , and . before departing to the ksa, participants ( . %) were positive for at least virus ( hrv, fluc, hadv, and hev), without coinfection. during the pilgrimage in the ksa, among the symptomatic pilgrims who were sampled, ( . %) were positive for at least virus ( hrv, flua, fluc, rsvb, hmpv, hadv, and hev), and there were double infections (flua/fluc, rsvb/hrv, and hrv/hev). this was significantly higher than the . % of pilgrims who were positive before departing to the ksa (p < . ). the prevalence of hrv was significantly higher during the hajj than before ( . % vs %; p < . ). of the pilgrims positive for hrv during their pilgrimage, were positive before traveling to the ksa. among the pilgrims ( . %) who met criteria for self-reported ili during the hajj, ( . %) were sampled, of whom ( . %) were virus-positive. the overall prevalence of respiratory viruses during the pilgrimage was significantly higher in pilgrims who reported more frequent hand-washing than usual during their stay compared to those who reported usual hand-washing ( . % vs . %; or, . ; % ci, . - . ; p = . ). no respiratory symptoms reported during the stay in the ksa were significantly associated with specific viral detection. before leaving the ksa, pilgrims ( %) were positive for at least virus ( hrv, hadv, flub, and hev), with double infections (hrv/hadv and hrv/hev), and this was significantly higher than the . % of pilgrims who were positive before departing to the ksa (p = . ). the prevalence of hrv was significantly higher before leaving the ksa than before departing to the ksa ( . % vs %; p = . ). of the pilgrims positive for hrv before leaving the ksa, were positive during their pilgrimage. the overall prevalence of respiratory viruses before leaving the ksa was significantly higher in individuals who reported using hand sanitizer during their stay compared to the remaining pilgrims ( . % vs . %; or, . ; % ci, . - . ; p = . ). all the pilgrims who were viruspositive before leaving the ksa complained of at least respiratory symptom during their stay. this is the first prospective longitudinal study investigating respiratory viruses from nasal specimens taken before departing for the ksa, during the pilgrimage in the ksa, and just before leaving the ksa in a single cohort of pilgrims, whether symptomatic or not. other studies have been conducted either among symptomatic pilgrims recruited in the ksa [ , ] , among separate populations of arriving and departing pilgrims [ ] , and in returned pilgrims only [ ] , or were limited to the influenza virus [ ] [ ] [ ] . nine pilgrims out of experienced respiratory symptoms during their stay in the ksa, with a cough and sore throat as the most common symptoms. ili symptoms were reported by . % of total pilgrims. an increase of respiratory symptoms was observed twice, following the tawaf, which is performed in the grand mosque in mecca in highly overcrowded conditions. an -fold increase in the overall prevalence of respiratory viruses was observed between samples obtained before departing from france ( . % of pilgrims) and samples obtained from ill pilgrims after performing their first tawaf in mecca ( . % of pilgrims). in our study, hrv was the most frequent virus detected from symptomatic pilgrims ( %). this result is in accordance with studies conducted worldwide where hrv has been recognized as the most frequent virus responsible for the common cold in adults [ , ] . this is not unexpected, as rhinoviruses are nonenveloped viruses that are more resistant in the environment than enveloped viruses. symptoms of hrv infection are generally mild and limited to the upper respiratory tract. in contrast, lower respiratory symptoms associated with hrv infection are prominent in patients who have underlying asthma or other chronic lung disease [ ] . in the present study, . % of pilgrims reported chronic respiratory diseases; therefore, prevention must be reinforced in this high-risk population. there have been arguments [ ] regarding whether rhinoviruses are spread chiefly from infected person to healthy individuals by direct contact to the fingers of healthy individuals by a handshake or indirect contact from the hands of an infected person to an intermediary surface [ ] , or through the aerosol route [ ] . our findings showed that more frequent hand-washing was significantly associated with feverishness and symptoms of ili and with a higher prevalence of respiratory viruses. however, these results may indicate a reverse causation, as pilgrims with symptoms or who believe themselves to be at greater risk may have washed their hands more frequently. a recent study found that hand disinfection did not reduce hrv infection or hrv-related common cold illnesses [ ] . the prevalence of hrv paralleled the increase of respiratory clinical symptoms, with a higher prevalence in pilgrims sampled in mecca city during the first week of the pilgrimage. influenza viruses ranked second after hrv among symptomatic pilgrims ( . %), and patients with influenza virus infection reported receiving a seasonal influenza vaccination prior to the hajj pilgrimage. other viruses were rarely found in our study. we previously reported that the novel coronavirus middle east respiratory syndrome (mers-cov; formerly known as hcov-emc) was not detected during the hajj season among the cohort pilgrims described here [ ] . other coronaviruses and parainfluenza viruses were not considered in this study and should be investigated in future studies. more than out of pilgrims tested positive for overall respiratory viruses just before leaving the ksa, which indicates a high potential for spreading viral pathogens into pilgrims' home countries. given that a significant proportion of french pilgrims also travel to their country of birth in north africa immediately after returning to france from the hajj [ ] , there is also a potential for spreading these viruses beyond france. the main limitation of this study is that, although the vast majority of pilgrims reported respiratory symptoms during their stay in ksa, the sampling by the medical doctor was limited to those who sought medical consultation from her. however, some symptomatic pilgrims were found to be negative for respiratory viruses. for those sampled at mina and medina, this was likely due to delayed medical consultation, as the specimen was collected several weeks after the onset of symptoms. second, symptom occurrence was collected retrospectively and was based on self-reporting; thus, the date of the medical consultation did not necessarily correspond to the selfreported date of the onset of respiratory symptoms. third, samples that were obtained at the beginning of the pilgrimage in the ksa were stored at room temperature ( °c) for periods up to days before being processed, which may have been resulted in the degradation of genetic material. this may have likely contributed to underestimating the frequencies of infection. alternative strategies to better preserve the samples must be considered, among which ethanol or nucleic acid lysis buffer [ ] could be used. fourth, a technical point is that hrv sequencing was not performed to determine how often new hrv infections were acquired during the stay in the ksa. finally, we cannot demonstrate that the viruses detected from nasal swabs were responsible for the symptoms, as nasal carriage in asymptomatic pilgrims was observed in some cases, and symptoms may have resulted from infection by other viruses [ ] , or possibly bacteria [ ] , that were not investigated in our study. although our results cannot be extrapolated to all pilgrims, our study illustrates the rapid acquisition of respiratory viruses among pilgrims during their stay in the ksa, particularly rhinovirus, and demonstrates the potential for spreading these infections to pilgrims' home countries upon their return. financial support. this work was supported by the marseille public hospitals authority (aorc). potential conflicts of interest. all authors: no reported conflicts. respiratory tract infections during the annual hajj: potential risks and mitigation strategies global public health implications of a mass gathering in mecca, saudi arabia during the midst of an influenza pandemic french ministry of foreign affairs. pilgrimage to mecca-organization hajj: journey of a lifetime health risks at the hajj respiratory tract infection during hajj pattern of admission to hospitals during muslim pilgrimage (hajj) causes of hospitalization of pilgrims in the hajj season of the islamic year influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination influenza and the hajj: defining influenza-like illness clinically simultaneous detection of influenza viruses a and b using real-time quantitative pcr pandemic a(h n ) influenza virus detection by real time rt-pcr: is viral quantification useful? applicability of a real-time quantitative pcr assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr a retrospective overview of enterovirus infection diagnosis and molecular epidemiology in the public hospitals of marseille rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than routine pcr tests influenza and respiratory syncytial virus infections in british hajj pilgrims viral respiratory infections at the hajj: comparison between uk and saudi pilgrims detection of respiratory viruses among pilgrims in saudi arabia during the time of a declared influenza a(h n ) pandemic viral etiology of acute respiratory infections among iranian hajj pilgrims influenza vaccine in hajj pilgrims: policy issues from field studies influenza viral infections among the iranian hajj pilgrims returning to shiraz, fars province pandemic influenza a (h n ) infection among hajj pilgrims from southern iran: a real-time rt-pcr-based study human rhinoviruses clinical effects of rhinovirus infections pathogenesis of rhinovirus infection transmission of rhinovirus colds by self-inoculation aerosol transmission of rhinovirus colds a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms travel reported by pilgrims from marseille evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification the viral etiology of an influenza-like illness during the pandemic bacteria and viruses that cause respiratory tract infections during the pilgrimage (haj) season in makkah, saudi arabia key: cord- -ckdx j authors: zheng, shou-yan; xiao, qiu-yan; xie, xiao-hong; deng, yu; ren, luo; tian, dai-yin; luo, zheng-xiu; luo, jian; fu, zhou; huang, ai-long; liu, en-mei title: association between secondary thrombocytosis and viral respiratory tract infections in children date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: ckdx j secondary thrombocytosis (st) is frequently observed in children with a variety of clinical conditions. the leading cause of st is respiratory tract infection (rti) in children. nasopharyngeal aspirate samples were collected and assessed for common respiratory viruses. the relationships between virus infections and secondary thrombocytosis were analyzed retrospectively. the blood platelet count and the presence of respiratory viruses were determined for rti patients, and ( . %) cases with platelet ≥ × ( )/l were considered as the thrombocytosis group. compared with the normal group, the detection rates of respiratory syncytial virus (rsv) and human rhinovirus (hrv) were significantly higher in the thrombocytosis group (p = . and . , respectively). hrv single infection was a risk factor associated with thrombocytosis [odds ratio (or) = . , % confidence interval (ci) = . – . ]. furthermore, st was more likely to occur in younger patients who had clinical manifestations of wheezing and dyspnea and who had been diagnosed with bronchiolitis. furthermore, the course of disease lasted longer in these patients. st is associated with viral respiratory tract infections, especially rsv and hrv infections. hrv single infection is a risk factor associated with thrombocytosis. scientific reports | : | doi: . /srep ethics statement. all experiments were approved by the ethics committee of the children's hospital of chongqing medical university. the guardians of the patients signed informed consent for participation in this study and for the publication of the individual clinical details. the methods were carried out in accordance with the approved guidelines. the study was conducted in compliance with the principles of the declaration of helsinki. study subjects and samples collection. children with respiratory tract infections (rtis) that were treated at the department of respiratory medicine at the children's hospital of chongqing medical university in china between june and may were enrolled in the study. in all patients, the diagnosis of rti was based on clinical, laboratorial and radiological evidence. nasopharyngeal aspirate (npa) samples were collected when the patients were admitted to our department. the specimens were kept at °c for a maximum of h and stored at − °c until further processing. the viral dna and rna were extracted from -μl aliquots of the npa samples using a qiaampminelute virus spin kit (qiagen, hilden, germany). the rna was applied as the template for cdna synthesis using the superscript iii first-strand synthesis system (invitrogen, california, usa). the dna and rna extractions and cdna products were used for subsequent testing for respiratory viruses. all of the samples were analyzed using a commercial detection kit (takara biotechnology, dalian, china and applied biosystems, california, usa), according to the manufacturer's instructions. multiplex nested polymerase chain reaction (pcr) was used to detect the following common respiratory viruses, as described previously [ ] [ ] [ ] [ ] [ ] : rsv subtypes a and b (rsva, rsvb); influenza virus (ifv) subtypes a, b and c (ifva, ifvb, ifvc); human coronaviruses (hcov); metapneumovirus (mpv); parainfluenza virus type to (piv - ); adenovirus (adv); human bocavirus type (hbov ) and human rhinovirus (hrv) subtypes a and c (hrva, hrvc). real-time pcr (rt-pcr) was also used to detect hrv and hbov . hrv-positive samples were further amplified and sequenced to identify the hrv subtype. platelet counting and thrombocytosis definition. complete blood counts were performed in the clinical laboratory using an xe- blood autoanalyzer (sysmex, japan). we used clinical features and published reference standards to define normal platelet counts as being between × /l and × /l for children. platelet counts of ≥ × /l were defined as thrombocytosis. moreover, to better consider the clinical characteristics and implications of thrombocytosis, patients with platelet counts between - × /l were diagnosed with mild thrombocytosis and those with counts of ≥ × /l were considered as moderate to severe thrombocytosis; platelet counts above × /l were considered indicative of extreme thrombocytosis . a retrospective review of the medical records was performed on all patients, and those who had at least one platelet count ≥ × /l during hospitalization were recruited into the thrombocytosis group. statistical analyses. statistical analyses were performed using the statistical package for the social sciences version . (spss . ). the categorical variables were compared using the chi-square test or fisher exact test, and the continuous variables were compared using student's t-test or the nonparametric mann-whitney u-test. correlation was analyzed using logistic regression analysis. p-values < . were considered to be significant. demographic data and platelet count of total patients. during the five-year study period, the blood platelet count and presence of respiratory viruses were randomly assessed in children ranging in age from month to years (median = months). approximately . % ( ) of these children were male. based on the clinical data, ( . %) cases with platelet counts ≥ × /l (median × /l) were considered as the thrombocytosis group, and ( . %) cases with platelet counts ranging between and × /l (median × /l) were considered as the normal group. among thrombocytotic patients, ( . %) with counts between and × /l were considered as having mild thrombocytosis, and ( . %) patients with counts of ≥ × /l were classified as having moderate to severe thrombocytosis; this last group included patients with platelet counts > × /l. thrombocytosis was presented in . % ( / ) of patients and most frequently ranged from to × /l. we observed that the platelet count began to elevate on approximately the th day of illness. the age distribution of patients with st was month to years (median months); most of these patients were younger than two years of age, ( . %), and were markedly younger than patients without st (median months) (p < . ). moreover, ( . %) of the thrombocytotic patients were male, but the gender distributions were not significantly different from the normal platelet patients. virus detection of the normal and thrombocytosis groups. the virus detection rates of the normal and thrombocytosis groups were . % ( / ) and . % ( / ), respectively. the single viral infection rates were . % ( / ) and . % ( / ), respectively, and the co-infection rates were . % ( / ) and . % ( / ), respectively. there was no significant difference in the total virus detection rate between the two groups. among the thrombocytosis group, rsv ( . %) was the most common virus detected, followed by hrv ( . %) and piv ( . %). the detection results for each virus and their different subtypes for both groups are summarized in table . compared with the normal group, the total detection rates of rsv and hrv were both significantly higher (p = . and . , respectively) in the thrombocytosis group, and adv and ifv were lower (p = . and . , respectively). the single viral infection rates of rsv and hrv in the thrombocytosis group were both significantly higher (p = . and . , respectively), and the single viral infection rates of adv and ifv in thrombocytosis group were significantly lower compared to the normal platelet group (p < . and p = . , respectively). however, the co-infection rate of viruses between two groups was not remarkably different except for rsv. the specific virus detection results are summarized in table . we further divided the thrombocytosis group into mild and moderate to severe groups and analyzed the viral infections of these subgroups. there was no marked difference of the virus detection rate between the two groups, with the exception that hbov co-infections were more common in the mild thrombocytosis group (tables and ). we also divided the normal platelet counts group into two subgroups: ( - ) × /l and ( - ) × /l, then analyzed the viral infections of the subgroups. the total detection rates of rsv and piv were both significantly higher (p = . and . , respectively) in the latter group, and adv and ifv were lower (p < . and . , respectively) ( table ) . clinical data in the normal and thrombocytosis groups. we compared the clinical conditions of the two groups, and the clinical manifestations of wheezing and dyspnea were significantly overrepresented in the thrombocytotic group patients compared to the normal group patients (p < . and p = . , respectively). however, the occurrence of fever in the normal group was remarkable higher than the thrombocytosis group. the most common clinical diagnoses of the two groups were lower respiratory tract infections, such as pneumonia, bronchiolitis, severe pneumonia, severe bronchiolitis and bronchitis. other conditions included upper respiratory tract infection, laryngitis, and amygdalitis. the incidence of bronchiolitis in the thrombocytosis group was significantly higher than in the normal group (p < . ). the days of illness before admission, length of hospitalization and course of disease in the thrombocytosis group were all longer compared with the normal group (p < . ). the c-reactive protein (crp) levels of ( . %) thrombocytotic patients were above mg/l. the percentage of patients with elevated crp levels was not significantly different between two groups, but the rate of anemia in the thrombocytosis group was higher (p = . ). in the thrombocytosis group, sputum culture-positive results were obtained in ( . %) cases, and virus detection and sputum cultures were both positive in ( . %) cases. the typical clinical findings are summarized in table . table . comparison of specific virus detection rate between the mild and moderate to severe thrombocytosis groups. moreover, we also investigated patients with very high platelet counts. there were patients with extreme thrombocytosis, defined as platelet counts of > × /l; the maximum count was × /l. of these patients, were less than one year old, were diagnosed with bronchiolitis, and were diagnosed with pneumonia. in of these cases, at least one viral infection was detected, and the primary viruses were rsva, piv , and hrva. the age distribution, occurrence of wheezing, prevalence of bronchiolitis, course of disease and viral infection were further analyzed using logistic regression models. in general, hrv single infection was a risk factor associated with thrombocytosis (or = . , % ci = . - . ) after considering the effects of age, course of the disease, occurrence of wheezing and diagnosis with bronchiolitis. hrva was especially strongly associated with thrombocytosis (or = . , % ci = . - . ) ( table ) . in the thrombocytosis group, no one developed thromboembolic or hemorrhagic complications. in total, ( . %) patients reached clinical recovery or improvement, ( . %) patients did not improve and ( . %) patients died of respiratory failure and septicemia. table . comparison of clinical data between the normal and thrombocytosis groups. *crp: c-reactive protein; crp level above mg/l were defined as increased. st is primarily found in pediatric patients with respiratory tract infections. in our study, st was identified in approximately . % ( ) of the inpatients with rti. st was especially common in patients with lower respiratory tract infections and occurred most frequently in the mild thrombocytosis group ( - × /l). previous studies showed that st occurs in - % of patients with respiratory tract infections [ ] [ ] [ ] . the different incidence rates may be explained by the cutoff platelet count, study population (inpatients, outpatients, or both), and median age of the enrolled subjects. additionally, the platelet count peaked on the th day after the onset of illness. this also supports the findings by other investigators who reported that platelet count in st peaked during the second and third week of the illness , . in our cases, the highest incidence of st occurred in patients who were under two years of age ( . %), which is consistent with the mastubara et al. study of japanese children with thrombocytosis, more than % of whom were aged less than two years . because bone marrow precursor cells of younger infants are more sensitive and rapid to external stimuli such as infections. our findings showed that childhood st is related to respiratory viral infections, but the degree of platelet elevation is unrelated to the species of virus. the total virus detection rate was . % ( / ) in patients with st. rsv ( . %) was the most commonly detected virus, followed by hrv ( . %) and piv ( . %). from the present data, we found that rsv single infection (especially subtype b) and hrv infection (especially subtype a) were prominent causative agents of thrombocytosis. to our knowledge, our study is the first to find that st occurrence is associated with hrv infection. in addition, hbov co-infection with other viruses in the mild thrombocytosis group is more common than in the moderate to severe thrombocytosis groups. this may be because this group of patients is frequently co-infected with rsv and hrv. according to the above results, we speculate that increased platelet counts may be indicative of a respiratory tract inflammatory reaction after rsv or hrv infection. the mechanism by which infection can promote thrombocytosis has not yet been fully elucidated. reports in the literature show that st is related to increased endogenous levels of several cytokines, such as thrombopoietin (tpo), interleukin- (il- ), interleukin- (il- ), interleukin- alpha (il- a) and tumor necrosis factor alpha (tnf-a) [ ] [ ] [ ] . it has been found that during rsv infection, many infants demonstrated rsv-specific cytokine responses, such as increased levels of il- and il- . however, st caused by hrv infection has not been described. bronchial epithelial cells secrete a wide variety of inflammatory cytokines, including il- , il- , il- , and gm-csf, and these cytokines are responsible for the development of thrombocytosis following rsv and hrv infections , . furthermore, tpo concentrations usually correlate with crp levels , . among our thrombocytotic patients, . % ( ) had elevated crp levels; this could indicate that crp participates in thrombocytosis. some authors have suggested that anemia may also play a role, and we observed a higher rate of anemia in the thrombocytosis group , . another theory confirmed that the number of megakaryocytes increased and that the pulmonary capillary bed released more platelets due to stimulation by tpo after infection. we believe the specific viral agents that cause rti induce inflammatory response and circulating cytokines that can then lead to st and perhaps other hematological disorders. patients that develop st have been more likely to also experience wheezing, dyspnea and a longer disease course. therefore, increased platelet counts may be a clinical marker associated with the severity of rti. our logistic regression analysis showed that patients who had clinical manifestations of wheezing, bronchiolitis and a longer course of disease were more inclined to develop thrombocytosis. additionally, hrv single infection was a risk factor associated with thrombocytosis. therefore, physicians should carefully monitor these patients. however, the mechanism through which this group of patients developed thrombocytosis has not been fully determined. the percentage of patients with fever in the normal group was higher than in the thrombocytosis group; this could possibly due to the presence of bacterial infections in patients with normal platelet counts. the great majority of our patients underwent clinical recovery or improvement. in childhood, st rarely results in thromboembolic or hemorrhagic complications, so treatment with platelet aggregation inhibitors is not required even when the platelet count is more than × /l. treatment should be targeted at the primary disease rather than the platelet count . we observed that the detection rate of adv and ifv was lower in children with st. generally, cytomegalovirus (cmv), epstein-barr virus (ebv) and parvoviridae often lead to thrombocytopenia , . one explanation for this observation could be that adv infection is more common in older children that do not frequently develop st. further work is needed to determine why adv and ifv seldom cause thrombocytosis. the limitations of our study include that the fact that this is a retrospective study rather than a cohort study, our study was performed in a single medical center, the subjects were limited to a young age group, and we had no follow-up study of platelet change. a large-scale cohort study is needed to investigate the causal relationship between viral infections and st in children. more specific studies are needed to further elucidate the inflammatory process involved in viral infection and the specific pathophysiology of thrombocytosis. in summary, research regarding viral respiratory tract infections and thrombocytosis is lacking, and we performed a large sample analysis that linked a specific viral agent to thrombocytosis in this clinical retrospective study. childhood st is related to respiratory tract infections, the occurrence rate is approximately . %, and the most common viruses are rsv and hrv, but infections with adv or ifv seldom cause thrombocytosis. hrv single infection is a risk factor associated with thrombocytosis. younger infants that are diagnosed with bronchiolitis and have a clinical manifestation of wheezing are more likely to develop st and undergo a longer course of disease. these observations suggest that increased platelet counts may be a retrospective marker of respiratory tract inflammatory reactions after viral infections and can indicate the severity of lower respiratory tract infections. aetiology and clinical significance of thrombocytosis: analysis of patients with an elevated platelet count guideline for investigation and management of adults and children presenting with a thrombocytosis reactive thrombocytosis in children with upper urinary tract infections primary and secondary thrombocytosis in childhood clinicohematological study of thrombocytosis in children thrombocytosis at an early stage of respiratory tract viral infection respiratory syncytial virus-positive bronchiolitis in hospitalized infants is associated with thrombocytosis thrombocytosis in pediatric patients is associated with severe lower respiratory tract inflammation incidence and clinical significance of reactive thrombocytosis in children aged to months, hospitalized for community-acquired infections simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay human bocavirus and acute wheezing in children use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types thrombocytosis and thrombocythemia thrombocytosis after pneumonia and empyema and other bacterial infections in children age-dependent changes in the incidence and etiology of childhood thrombocytosis circulating levels of thrombopoietic and inflammatory cytokines in patients with clonal and reactive thrombocytosis stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand interleukin- stimulates thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis human infant respiratory syncytial virus (rsv)-specific type and cytokine responses ex vivo during primary rsv infection host immune responses to rhinovirus: mechanisms in asthma rhinovirus induces airway epithelial gene expression through double-stranded rna and ifn-dependent pathways elevation of serum thrombopoietin precedes thrombocytosis in acute infections clinicohematological study of thrombocytosis screening children with thrombosis for thrombophilic proteins. cui bono? virus-associated immune thrombocytopenic purpura in childhood virus-associated idiopathic thrombocytopenic purpura we acknowledge the assistance of the patients and their caregivers involved in the study, the staff of the e.m.l. and a.l.h. conceived and designed this study and revised the manuscripts. s.y.z., q.y.x., x.h.x., y.d., l.r., d.y.t., z.x.l., j.l. and z.f. collected the samples and performed the experiments. s.y.z. and q.y.x. analyzed the data and wrote the paper. all authors reviewed the manuscript. all authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.