key: cord- -skavefji authors: choi, sang-ho; hong, sang-bum; hong, hyo-lim; kim, sung-han; huh, jin won; sung, heungsup; lee, sang-oh; kim, mi-na; jeong, jin-yong; lim, chae-man; kim, yang soo; woo, jun hee; koh, younsuck title: usefulness of cellular analysis of bronchoalveolar lavage fluid for predicting the etiology of pneumonia in critically ill patients date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: skavefji background: the usefulness of bronchoalveolar lavage (bal) fluid cellular analysis in pneumonia has not been adequately evaluated. this study investigated the ability of cellular analysis of bal fluid to differentially diagnose bacterial pneumonia from viral pneumonia in adult patients who are admitted to intensive care unit. methods: bal fluid cellular analysis was evaluated in adult patients who underwent bronchoscopic bal following less than hours of antimicrobial agent exposure. the abilities of bal fluid total white blood cell (wbc) counts and differential cell counts to differentiate between bacterial and viral pneumonia were evaluated using receiver operating characteristic (roc) curve analysis. results: bacterial pneumonia (n = ) and viral pneumonia (n = ) were frequently associated with neutrophilic pleocytosis in bal fluid. bal fluid median total wbc count ( , /µl vs. /µl, p< . ) and percentage of neutrophils ( . % vs. . %, p = . ) were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. in roc curve analysis, bal fluid total wbc count showed the best discrimination, with an area under the curve of . ( % ci, . – . ). bal fluid total wbc count ≥ /µl had a sensitivity of . %, specificity of . %, positive likelihood ratio (plr) of . , and negative likelihood ratio (nlr) of . . when analyzed in combination with serum procalcitonin or c-reactive protein, sensitivity was . %, specificity was . %, plr was . , and nlr was . . bal fluid total wbc count ≥ /µl was an independent predictor of bacterial pneumonia with an adjusted odds ratio of . in multiple logistic regression analysis. conclusions: cellular analysis of bal fluid can aid early differential diagnosis of bacterial pneumonia from viral pneumonia in critically ill patients. severe pneumonia requiring intensive care unit (icu) admission is associated with high rates of morbidity and mortality. delays in the provision of adequate antimicrobial therapy have been reported to be associated with excess mortality [ ] [ ] [ ] ; therefore, rapid and accurate etiologic diagnosis of severe pneumonia is essential for successful treatment. in recent years, bronchoscopic bronchoalveolar lavage (bal) has been established as a useful technique for collecting lower respiratory tract specimens from the alveolar level, and can thus be used to accurately define the causative organisms of pneumonia [ ] [ ] [ ] . however, a conventional culture usually takes at least a few days, and microbiological yield is often compromised by prior empirical usage of antimicrobial agents. in addition, identification of viruses and atypical organisms requires a separate etiologic work-up. cellular analysis of bal fluid, including total and differential cell counts and the cd +:cd + t-lymphocyte ratio, is useful for the diagnosis of various interstitial lung diseases [ ] [ ] [ ] . under an appropriate clinical setting, bal fluid analysis can provide highly suggestive or even diagnostic information for specific interstitial lung diseases in the absence of a lung biopsy [ ] . however, only a few previous studies with limited patient populations [ ] [ ] [ ] have evaluated the role of cellular analysis of bal fluid in patients with suspected pneumonia. most of these studies focused on the differential diagnosis of pneumonia from non-infectious pulmonary diseases, not on the prediction of pneumonia etiology. bal fluid analysis can be performed within several hours. therefore, such analysis would be useful for guiding early treatment if it could predict the etiology of pneumonia, similar to the role of cerebrospinal fluid cellular analysis, which can reliably differentiate among meningitis etiologies. therefore, this study investigated whether analysis of the cellular profile of bal fluid can predict the etiology of pneumonia in critically ill patients admitted to the medical icu. this study was based on data from a prospective observational cohort study conducted from march to may . all patients admitted to the medical icu of asan medical center, a , -bed tertiary care university-affiliated hospital in seoul, republic of korea, with suspected severe pneumonia were prospectively identified and monitored until discharged [ ] . the data collected included patient demographics, underlying diseases or conditions, illness severity scores including acute physiological and chronic health evaluation (apache) ii and sequential organ failure assessment (sofa), type of pneumonia, laboratory data including microbiological tests, length of icu stay, and outcome. the prospectively collected data were retrospectively analyzed. this study was approved by the institutional review board of asan medical center and the requirement for informed consent was waived because of the observational nature of the study. all patients information was anonymized and deidentified prior to analysis. inclusion criteria were as follows: ( ) patients aged $ years with a clinical diagnosis of pneumonia (see below for definition), and ( ) patients who underwent bronchoscopic bal for etiologic diagnosis of pneumonia. exclusion criteria were as follows: ( ) patients in whom the pathogen was not identified, ( ) patients in whom bal fluid analysis was impossible (due to severe neutropenia or clotting of specimen) or not performed, ( ) patients with a mixed infection (identification of bacteria and virus), ( ) patients who were treated with antimicrobial agents for more than hours before bronchoscopic bal, ( ) patients with invasive pulmonary aspergillosis, ( ) patients with mycobacterial infection, and ( ) patients with pneumocystis jirovecii pneumonia. pneumonia was defined as the presence of an acute infiltrate on a chest radiograph and at least one of the following: fever (temperature $ . uc) or hypothermia (temperature , . uc), cough, pleuritic chest pain, dyspnea, and altered breath sounds on auscultation [ ] . pneumonia was categorized as communityacquired pneumonia (cap), healthcare-associated pneumonia (hcap), or hospital-acquired pneumonia (hap), as defined previously [ , ] . fiberoptic bronchoscopy with bal was performed following a standardized protocol as previously described [ ] . briefly, bal was performed by instillation of three consecutive aliquots of sterile saline solution ( - - ml) into the bronchial tree at the area that was most abnormal on the chest radiography. the right middle lobe or lingual segment was chosen in patients with bilateral diffuse infiltration. bal fluid that was first retrieved was discarded, and bal fluid that was subsequently retrieved was collected. the total cell count was determined using a hemocytometer. the corresponding amount of bal fluid for cells was centrifuged onto a microscope slide using a thermo shandon cytospin (thermo fisher scientific inc., waltham, ma, usa), at rpm for minutes at room temperature. the slide was airdried and stained with wright-giemsa stain. differential cell counts that included percentages of neutrophils, lymphocytes, alveolar macrophages, and eosinophils were determined. bacterial, fungal, and mycobacterial cultures of endotracheal aspirates and bal fluid were performed. respiratory viruses were tested by a multiplex reverse-transcription polymerase chain reaction (pcr) assay using a seeplex rv ace detection kit (seegene inc., seoul, korea) and/or shell vial culture. pcr to detect mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila, and a urinary antigen test to detect streptococcus pneumoniae and l. pneumophilia serogroup species were also performed. data were expressed as mean standard deviation or median and - % interquartile range according to data distribution. categorical variables were compared using the chi-square test or fisher's exact test as appropriate. receiver operating characteristic (roc) curves were constructed to determine the performances of bal fluid cellular components, serum procalcitonin concentration, and c-reactive protein concentration for predicting bacterial pneumonia. youden's index (sensitivity + specificity- ) [ ] was used to select the optimal cutoff points of the roc curve. area under the curve (auc), sensitivity, specificity, positive likelihood ratio and negative likelihood ratio were calculated. for positiveand negative predictive values, the prevalence of bacterial pneumonia in severe pneumonia patients admitted to the medical icu was assumed to be . %, based on our previous study [ ] . multivariable logistic regression analysis was used to identify independent predictors of bacterial pneumonia. variables with p values less than . in the univariate analysis were included in the multivariate analysis. the correlation between bal fluid white blood cell (wbc) count and apache ii score was determined by calculating pearson's correlation coefficient. significance was accepted at p # . . all tests were performed using spss (version . ; spss, inc.) and graphpad prism (version ; graphpad, inc.) software. pneumonia underwent bronchoscopic bal ( with cap, with hcap, and with hap). of these patients, were excluded because the pathogen was not identified, were excluded because bal fluid analysis was not possible (due to severe neutropenia or specimen clotting) or not performed, were excluded because two or more types of pathogens were identified, and were excluded because they received antimicrobial therapy for more than hours before bronchoscopic bal. ten patients with pneumocystis jirovecii pneumonia, patients with invasive pulmonary aspergillosis, and patients with mycobacterial pneumonia were also excluded. finally, patients ( with bacterial pneumonia and with viral pneumonia) were included. the characteristics of the patients are shown in table . thirty-two patients ( . %) were men and the mean age was . years. structural lung disease was the most common underlying disease ( . %), followed by diabetes mellitus ( . %), and hematologic malignancy/solid cancer (both . %). sixteen patients ( . %) had cap, ( . %) had hcap, and ( . %) had hap. most baseline characteristics did not significantly differ between the bacterial pneumonia and viral pneumonia groups. by contrast, mean apache ii ( . . vs. . . , p = . ) and sofa ( . . vs. . . , p = . ) scores were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. however, mortality rates, including -day mortality, did not significantly differ between the groups. pathogens that were identified in patients are summarized in table . twenty-eight bacterial pathogens were identified in patients. in patients, two different bacteria were identified. staphylococcus aureus (n = ) was the most common bacteria, followed by legionella pneumophila (n = ), and streptococcus pneumoniae (n = ). bacteria were identified from bal fluid cultures or pcrs in patients, from endotracheal aspirates or sputum cultures in patients, from blood cultures in patients, and from urinary antigen tests in patients (two patients with pneumococcal antigens and two patients with legionella antigens). eleven patients had two or more positive tests. twenty-six viruses were identified in patients. in three patients, two different viruses were identified. rhinovirus was the most common virus (n = ), followed by influenza virus (n = ), and respiratory syncytial virus (n = ). viruses were identified from bal fluid specimens in patients and from nasopharyngeal specimens in patients. viruses were detected in both in bal fluid and nasopharyngeal samples in patients. cellular bal fluid profiles and distributions of bal fluid cell counts in the two groups are shown in table and figure . detailed data of each patient are summarized in table s . the median total wbc count ( , /ml vs. /ml, p, . ), percentage of neutrophils ( . % vs. . %, p = . ), and absolute neutrophil count ( , /ml vs. /ml, p, . ) of bal fluid were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. the median serum procalcitonin concentration was also higher in the bacterial pneumonia group than in the viral pneumonia group ( . ng/ml vs. . ng/ml, p = . ), and the c-reactive protein concentration tended to be higher in the bacterial pneumonia group than in the viral pneumonia group ( . mg/dl vs. . mg/dl, p = . ). of the pathogen-identified patients who had received antimicrobial agent for more than hours prior to bronchoscopic bal (figure ) , had bacterial pneumonia, had viral pneumonia, and had invasive pulmonary aspergillosis. of the patients with bacterial pneumonia or viral pneumonia, the median duration of antimicrobial therapy before bronchoscopic bal was days (interquartile range, - days). the median total wbc count ( /ml vs. /ml, p = . ) and percentage of neutrophils ( . % vs. . %, p = . ) were not significantly different between these two groups. figure s shows the changes recorded in the bal fluid total wbc count and percentage of neutrophils according to the duration of exposure to antimicrobial agents. diagnostic performances of bal fluid cellular components, serum procalcitonin concentration, and serum c-reactive protein concentration for the prediction of bacterial pneumonia the ability of bal fluid cellular analysis to distinguish between bacterial pneumonia and viral pneumonia was assessed using roc analysis (table last the sensitivities, specificities, positive predictive values, negative predictive values, positive likelihood ratios, and negative likelihood ratios are summarized in table . when the cutoff value of bal fluid total wbc count was $ /ml, which was selected using youden's index, sensitivity was . % ( % ci; . - . ), specificity was . % ( % ci; . - . ), positive predictive value was . % ( % ci; . - . ), negative predictive value was . % ( % ci; . - . ), positive likelihood ratio was . ( % ci; . - . ), and negative likelihood ratio was . ( % ci; . - . ). a combination of bal fluid total wbc count $ /ml or serum procalcitonin concentration $ . ng/ml had a sensitivity of . % ( % ci; . - . ) and a negative likelihood ratio of . ( % ci; . - . ), whereas bal fluid total wbc count $ /ml and serum c-reactive protein concentration $ . mg/dl had specificity of . % ( % ci, . - . ) and a positive likelihood ratio of . ( % ci, . - . ). when a cutoff value of bal fluid total wbc count $ /ml was applied to pathogen-identified patients who had received antimicrobial agent for more than hours, sensitivity was . % ( % ci; . - . ), specificity was . % ( % ci; . - . ), positive predictive value was . % ( % ci; . - . ), negative predictive value was . % ( % ci; . - . ), positive likelihood ratio was . ( % ci; . - . ), and negative likelihood ratio was . ( % ci; . - . ). multiple logistic regression analysis revealed that bal fluid total wbc count $ /ml was an independent predictor of bacterial pneumonia with an adjusted odds ratio of . ( % ci; . - . ) ( table ). there was a modest but significant positive correlation between the degree of bal leukocytosis and the apache ii score (r = . , p = . ) (figure ) . table . bronchoalveolar lavage total and differential cell counts (%) in patients with pneumonia. this study analyzed the usefulness of cellular analysis of bal fluid for predicting the etiology of pneumonia in critically ill adult patients. neutrophilic pleocytosis in bal fluid was frequently found in patients with bacterial-and viral pneumonia. the degree of pleocytosis, which was higher in the bacterial pneumonia, was useful for differential diagnosis of bacterial pneumonia. total wbc count had the best diagnostic accuracy for predicting bacterial pneumonia, and its diagnostic performances was better than those of serum procalcitonin and c-reactive protein concentrations. combinations of bal fluid total wbc count, serum procalcitonin concentration, and serum c-reactive protein concentration provided the best diagnostic yields. the data suggest that cellular analysis of bal fluid is a rapid and useful technique for differentiating bacterial pneumonia from viral pneumonia, and can be used to direct early appropriate treatment. information about the role of cellular profiles of bal fluid for differential diagnosis of bacterial pneumonia in adult patients is limited. stolz et al [ ] . evaluated potential markers of bacterial infection in a cohort of immunocompromised patients with pulmonary complications. they reported that the percentage of neutrophils in bal fluid and the serum procalcitonin concentration are independent predictors of bacterial infection. they suggested that the optimal cutoff value of the percentage of neutrophils in bal fluid is % (sensitivity %; specificity %), which is much lower than the cutoff value in the current study. sternberg et al. [ ] investigated the usefulness of bal in assessing pneumonia in renal transplant patients, and suggested that the optimal cutoff value of the percentage of neutrophils in bal fluid is . % for predicting bacterial pneumonia. however, neither of these previous studies included patients with severe pneumonia caused by respiratory viruses alone, and both compared bal findings between patients with bacterial pneumonia and those with non-infectious diseases. in the current study, patients with viral pneumonia were included by using the newly developed multiplex respiratory virus rt-pcr. this showed that cases of viral pneumonia were frequently associated with neutrophilia in bal fluid (median . %). we speculate that this underlies why the optimal cutoff value of percentage of neutrophils in bal fluid for predicting bacterial pneumonia is much higher in the current study ( %, table ) than in previous studies. several authors of the current study previously investigated the diagnostic utility of soluble triggering receptor expressed on myeloid cells- (strem- ) in bal fluid of various patient populations with bilateral lung infiltrates. a cutoff value of $ % neutrophils in bal fluid is useful for differential diagnosis of bacterial or fungal pneumonia from other causes of pneumonia or non-infectious diseases (auc = . , % ci; . - . , p = . ) [ ] . in comparison to this previous study, the current study did not include patients with non-infectious diseases or fungal pneumonia, included much more cases of severe viral pneumonia, and analyzed the counts of various cell types. among the currently available inflammatory markers, serum procalcitonin is one of the best indicators of bacterial infections, including lower respiratory tract infections [ ] . the usefulness of serum procalcitonin measurements has been validated in the diagnosis, severity assessment, and follow-up of patients with lower respiratory tract infections [ ] [ ] [ ] . in the current study, the auc of serum procalcitonin concentration for predicting bacterial pneumonia (auc = . ) was smaller than those of total wbc (auc = . ) and neutrophil (auc = . ) counts. the combination of bal fluid wbc counts and serum procalcitonin concentration tended to improve the diagnostic accuracy of the roc model. this indicates that combinations of these markers can be useful to rule-out (bal fluid total wbc count $ /ml or serum procalcitonin concentration $ . ng/ml with a sensitivity of . % and negative likelihood ratio of . ) or rule-in (bal fluid total wbc count $ /ml and serum c-reactive protein concentration $ . mg/dl with a specificity of . % and positive likelihood ratio of . ) bacterial pneumonia. diagnostic accuracy could be further improved if bal fluid cellular profiles are interpreted alongside clinical presentations, radiographic studies, and other relevant test results. using this approach, it might be possible to identify patients who can be managed without antibacterial agents or those who require antiviral agents. although not included in the current study, strem- in bal fluid is another notable biomarker for the diagnosis of pneumonia. strem- is reportedly a potent discriminator of bacterial pneumonia from non-infectious lung infiltrations [ , [ ] [ ] [ ] [ ] [ ] . however, the proposed cutoff values of strem- concentration vary widely ( - pg/ml) and some studies have questioned the reliability of bal fluid strem- [ ] [ ] [ ] . studies on bal fluid strem- have mainly been confined to patients with ventilator-associated pneumonia. therefore, the usefulness of strem- for etiologic diagnosis of pneumonia, especially differential diagnosis of viral pneumonia, has not been elucidated yet. the current study directly compared patients with bacterial and viral pneumonia, and therefore differs from previous studies of strem- . bal fluid strem- concentration in combination with the bal fluid cellular profile might exhibit better diagnostic performance, although this warrants further studies. a strength of the current study is the relatively strict enrollment criteria used. to minimize bias associated with antimicrobial therapy, all patients who received antimicrobial therapy for more than hours were excluded, regardless of the adequacy of prior antimicrobial therapy. by using strict enrollment criteria, however, only a small proportion of pneumonia patients who underwent bronchoscopic bal was finally included (figure ), which might have influenced on the results. however, positive likelihood and negative likelihood ratio, which are not influenced by disease prevalence, were good, which supports the validity of the data. in clinical practice, the results might be applied to patients who have received antimicrobial therapy more than hours. that is, if bal fluid cellular analysis shows evident pleocytosis even after antimicrobial therapy for more than hours, it would be a strong suggestion for bacterial etiology. the study has several limitations. first, the small sample size of the select critically ill patient population analyzed limits the general applicability of our findings. moreover, since our study included critically ill patients with acute respiratory failure secondary to pneumonia who were not receiving antimicrobial therapy, our results may not be applicable to the majority of severe pneumonia patients in clinical practice. second, the impact of antimicrobial therapy on the both bal fluid cellular profiles and other inflammation markers such as procalcitonin, remains to be further elucidated. third, cases of invasive pulmonary aspergillosis, pneumocystis jirovecii pneumonia, and mycobacterial pneumonia, were not included, mainly because few patients had these types of pneumonia. fourth, patients with non-infectious causes of pulmonary infiltrates that can often mimic infectious causes, such as acute respiratory distress syndrome, cryptogenic organizing pneumonia, eosinophilic pneumonia, and drug-induced pneumonitis, were also excluded from our analyses. the inclusion of those cases may have caused a marked decrease in the specificity of our bal fluid criteria. finally, all the pathogens were not directly identified from bal fluid. some patients were included in whom pathogens were identified by other means, such as blood culture, endotracheal aspirates culture, urinary pneumococcal antigen test, and pcr from nasopharyngeal samples, as long as clinically and radiographically compatible and no other etiology was demonstrated. therefore, patients with coincidental upper respiratory infections or colonization may have been included. in conclusion, the data indicate that cellular analysis of bal fluid, alone or in combination with serum procalcitonin and creactive protein concentrations, may rapidly provide valuable diagnostic information for the early differential diagnosis of pneumonia in critically ill adult patients. timing of antibiotic administration and outcomes for medicare patients hospitalized with community-acquired pneumonia inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients prognostic factors of non-hiv immunocompromised patients with pulmonary infiltrates quantitative culture of bronchoalveolar lavage fluid for the diagnosis of bacterial pneumonia diagnostic accuracy of bronchoalveolar lavage samples in immunosuppressed patients with suspected pneumonia: analysis of a protocol bronchoalveolar lavage for diagnosing acute bacterial pneumonia interstitial lung disease guideline: the british thoracic society in collaboration with the thoracic society of australia and new zealand and the irish thoracic society an official american thoracic society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease bronchoalveolar lavage cell populations in the diagnosis of sarcoidosis bronchoalveolar lavage: a forgotten tool bal neutrophils, serum procalcitonin, and c-reactive protein to predict bacterial infection in the immunocompromised host diagnostic utility of the soluble triggering receptor expressed on myeloid cells- in bronchoalveolar lavage fluid from patients with bilateral lung infiltrates utility of bronchoalveolar lavage in assessing pneumonia in immunosuppressed renal transplant recipients viral infection in patients with severe pneumonia requiring intensive care unit admission health care-associated pneumonia requiring hospital admission: epidemiology, antibiotic therapy, and clinical outcomes infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia optimal cut-point and its corresponding youden index to discriminate individuals using pooled blood samples procalcitonin in bacterial infections-hype, hope, more or less usefulness of procalcitonin levels in community-acquired pneumonia according to the patients outcome research team pneumonia severity index effect of procalcitonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial pneumonitis-associated hyperprocalcitoninemia soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia combined measurement of procalcitonin and soluble trem- in the diagnosis of nosocomial sepsis triggering receptors expressed on myeloid cells in pulmonary aspiration syndromes diagnostic implications of soluble triggering receptor expressed on myeloid cells- in patients with acute respiratory distress syndrome and abdominal diseases: a preliminary observational study diagnostic implications of soluble triggering receptor expressed on myeloid cells- in bal fluid of patients with pulmonary infiltrates in the icu soluble triggering receptor expressed on myeloid cells- in bronchoalveolar lavage fluid is not predictive for ventilator-associated pneumonia soluble triggering receptor expressed on myeloid cells- (strem- ) as a diagnostic marker of ventilator-associated pneumonia soluble triggering receptor expressed on myeloid cell- is increased in patients with ventilator-associated pneumonia: a preliminary report key: cord- -rft vo authors: rohde, g.; message, s. d.; haas, j. j.; kebadze, t.; parker, h.; laza‐stanca, v.; khaitov, m. r.; kon, o. m.; stanciu, l. a.; mallia, p.; edwards, m. r.; johnston, s. l. title: cxc chemokines and antimicrobial peptides in rhinovirus‐induced experimental asthma exacerbations date: - - journal: clin exp allergy doi: . /cea. sha: doc_id: cord_uid: rft vo rationale: rhinoviruses (rvs) are the major triggers of asthma exacerbations. we have shown previously that lower respiratory tract symptoms, airflow obstruction, and neutrophilic airway inflammation were increased in experimental rv‐induced asthma exacerbations. objectives: we hypothesized that neutrophil‐related cxc chemokines and antimicrobial peptides are increased and related to clinical, virologic, and pathologic outcomes in rv‐induced exacerbations of asthma. methods: protein levels of antimicrobial peptides (slpi, hnp – , elafin, and ll‐ ) and neutrophil chemokines (cxcl /gro‐α, cxcl /gro‐β, cxcl /ena‐ , cxcl /gcp‐ , cxcl /nap‐ , and cxcl /il‐ ) were determined in bronchoalveolar lavage (bal) fluid of asthmatics and normal controls taken before, at day four during and weeks post‐experimental infection. results: bal hnp – and elafin were higher, cxcl /nap‐ was lower in asthmatics compared with controls at day (p = . , p = . , and p = . , respectively). bal hnp – and cxcl /il‐ were increased during infection (p = . and p = . , respectively). there was a trend to increased bal neutrophils at day compared with baseline (p = . ). bal hnp – was positively correlated with bal neutrophil numbers at day . there were no correlations between clinical parameters and hnp – or il‐ levels. conclusions: we propose that rv infection in asthma leads to increased release of cxcl /il‐ , attracting neutrophils into the airways where they release hnp – , which further enhances airway neutrophilia. strategies to inhibit cxcl /il‐ may be useful in treatment of virus‐induced asthma exacerbations. patients with atopic asthma are more susceptible to lower respiratory tract (lrt) infections and have more severe and longer-lasting rhinovirus (rv)-induced lrt symptoms than healthy individuals [ ] . virus infections of the respiratory tract are frequently associated with asthma exacerbations, with rvs as the predominant viruses [ , ] . rvs directly infect the lower airways [ ] resulting in increased lower respiratory symptoms, reductions in lung function, bronchial inflammation, and augmented airway hyperresponsiveness in asthmatic compared with normal subjects [ ] . neutrophils are major effector cells in defence against invading pathogens [ ] , and their number has been shown to increase during rv infection in both experi-mental models [ , ] and naturally occurring asthma exacerbations [ ] . antimicrobial peptides of the defensin or cathelicidin families comprise a significant part of the neutrophilic armamentarium against these pathogens [ ] . the a-defensins (hnp - ) are stored in primary (azurophil) neutrophil granules and constitute - % of the total protein of these organelles [ ] . it has been hypothesized that human rhinovirus infections should increase levels of a-defensins in the airways [ ] , as they lead to marked neutrophil infiltration and degranulation in the airways [ ] which are associated with clinical severity of virus-induced asthma [ , ] . however, there have been no reports directly measuring defensins in the airways of subjects with virus-induced asthma so far. the human cathelicidin ll- is also released by neutrophils upon inflammatory stimulation and has potent bactericidal activity [ ] . slpi is another antimicrobial peptide produced by neutrophils (also by alveolar macrophages and epithelial cells) which may play a role in acute exacerbations of asthma. their role in virus-induced asthma is unknown. neutrophils are attracted to the airways and are activated mainly by the cxc chemokines cxcl /gro-a, cxcl /gro-b, cxcl /ena- , cxcl /gcp- , cxcl / nap- , and cxcl /il- . some of these (cxcl /gro-a, cxcl /gro-b, and cxcl /gcp- ) also have antimicrobial properties, while it has also been shown that elafin, another antimicrobial peptide expressed by alveolar macrophages and epithelial cells, is also chemotactic for neutrophils [ ] . against this background, we hypothesized that antimicrobial peptides are induced by rv infections in the lower airways in vivo. to test this hypothesis and to clarify whether this possible induction is related to airway neutrophilia and the expression of cxc chemokines, we analysed the expression of neutrophil antimicrobial peptides and cxc chemokines in bal fluid of subjects with rv-induced experimental asthma exacerbations. some of the results of this study have been previously reported in abstract form [ ] . the study design and the clinical and lower airway inflammation data of the patients investigated have been recently published in detail [ ] . briefly, two different groups were studied. the first group consisted of outpatients with mild atopic asthma; the second group were healthy non-atopic individuals. clinical and atopic status were defined by questionnaire, skin prick testing, serum ige, and lung function testing including pef, forced expiratory volume in s (fev ), forced vital capacity (fvc), and histamine challenge performed according to guidelines [ ] . the asthmatic group had a concentration of histamine causing a % reduction in fev (pc ) < mg/ml, the normal group > mg/ml. normal subjects were taking no medication; asthmatics inhaled short-acting b -agonists only. none of the asthmatic patients were given any inhaled or oral/systemic steroid at any time point in the study. subjects were free of common cold symptoms for weeks before commencing the study. all were non-smokers. bronchoalveolar lavage (bal) sampling was carried out at baseline ( weeks prior to virus inoculation), on day after inoculation (acute infection) and at weeks after inoculation (convalescent). diaries were kept to record symptoms and home lung function throughout the study. all subjects gave written informed consent, and the study was approved by the st mary's research ethics committee, st mary's hospital, london, uk. all subjects were seronegative (neutralizing antibody titre < : ) for rv at screening and on repeat serology performed on day prior to inoculation, and all subjects were negative to a pcr panel for respiratory viruses (adenoviruses, coronaviruses e and oc , human metapneumovirus, influenza ah /ah /b, other picornaviruses, parainfluenza viruses - , and respiratory syncytial virus) and mycoplasma and chlamydophila pneumoniae in nasal lavage at baseline [ ] . experimental infection was induced using tcid rv [ ] on day , with a devillbiss atomizer as described [ ] . following inoculation, subjects returned home. clinical effects of rv infection were recorded using daily diary cards enabling the calculation of a peak cold score, a total cold score (total over the week period post-inoculation), peak and total chest scores (all corrected for baseline symptoms and effect of bronchoscopy), lung function testing by home spirometry (mic-rodl, micromedical, carefusion, basingstoke, uk), and histamine challenge were performed as described [ ] . bronchoalveolar lavage was collected in a single plastic chamber and transferred immediately to polypropylene tubes on ice for transport to the laboratory. an aliquot of bal fluid was stored unprocessed at À °c for analysis of virus load by pcr. the remaining bal fluid was centrifuged, and bal fluid was stored in aliquots at À °c. the bal cell pellet was used for cytospin preparations for differential cell counting as described [ ] . rhinovirus infection was confirmed in all subjects using pcr, by culture or by serology as described [ ] . virus load was determined in nasal lavage and the unprocessed bal aliquot by quantitative pcr as described [ ] . in bal fluid, slpi levels were assessed by enzyme-linked immunosorbent assay (elisa) using a commercially available kit (r&d systems, abingdon, uk) with a sensitivity of < pg/ml. samples were diluted : . hnp - levels were measured by elisa, using a commercially available kit (hycult biotechnology, uden, the netherlands) with a sensitivity of < pg/ml. samples were diluted : . elafin and human ll- were assessed by elisa kits from cambridge bioscience, uk, with sensitivities of < pg/ml and < . ng/ml, respectively. cxcl /il- , cxcl /ena- , and cxcl /gro-a levels in bal fluid were assessed by luminex analysis (biosource) on the luminex tm system with sensitivities of < , < , and < pg/ml, respectively. cxcl / gcp- and cxcl /nap- were analysed by elisa using commercially available kits (r&d systems) with sensitivities of < . pg/ml as well as cxcl /gro-b (antigenix america inc, huntington station, ny, usa) with a sensitivity of < pg/ml. all data were checked for normal distribution by kolmogorov-smirnov test. normally distributed data are presented as mean and standard deviation, whereas non-normally distributed data are presented as median and interquartile range. differences between normal and asthmatic groups were analysed using unpaired t-tests for normally distributed data and mann-whitney test for non-normally distributed data. for discrete variables, frequencies were reported and compared by chi-square test or fisher's exact test as appropriate. the yates correction procedure was applied to all comparisons. differences during infection from baseline and convalescence were analysed using one-way repeated-measures anova for normally distributed data. sphericity was assessed by mauchly's test. if the assumption of sphericity was violated, degrees of freedom were corrected using greenhouse-geisser correction for e < . or huynh-feldt correction for e > . , respectively. in the case of significant differences, post hoc tests (bonferroni correction) were performed. in case of nonnormally distributed data, friedman's test was used and, if significant, post hoc tests (wilcoxon) were performed. correlations for normally distributed variables were examined using pearson's correlation coefficient, for non-normally distributed variables using spearman's correlation coefficient, and the respective two-tailed significance was reported. all significance levels were set to %. data were analysed and processed using graphpad prism . (graphpad software, inc., la jolla, ca, usa) and spss . (international business machines corp., armonk, ny, usa). the study design, the analysis of clinical characteristics and the clinical response to experimental viral infection together with extensive data on the effect on the th / th immune response have been reported [ ] . however, here, we present a completely new analysis of data from those atopic asthmatics and non-atopic normal controls that entirely completed the study. baseline characteristics of all recruited subjects ( asthmatics and controls) have been reported by message et al. [ ] recently. one asthmatic and two normal volunteers did not continue after the baseline phase. the clinical characteristics of the subjects that completed the study and who underwent the chemokine and anti-microbial peptide analyses reported in the present study are summarized in table . there were no significant differences between groups concerning age, gender, and baseline fev . features of allergic sensitization were only expressed in the asthmatic group. we reported before that asthmatic patients showed significantly higher total chest symptom score, significantly higher maximum falls in fev and pef, and significantly lower pc values at baseline, day , and week compared with healthy controls [ ] . lung function impairment induced by rv infection was correlated with increased neutrophils in bal of asthmatics suggesting a role for pmns in rv-induced exacerbations of asthma [ ] . there were no significant differences in virus load in upper and lower airway samples between the two groups. results are summarized in table . to determine differences in mediator release between normal and asthmatics subjects before, during, and after rv infection, a univariate analysis between groups was performed. this showed that bal cxcl /il- was the only parameter significantly different at baseline. interestingly, it was higher in the control group compared with asthmatics ( . ( . ) vs. . ( . ) pg/ml, p = . , fig. ). four days after infection, bal hnp - and elafin were significantly higher in asthmatics compared with controls repeated-measures multivariate analysis showed significant differences only in asthmatic subjects. bal hnp - and cxcl /il- were significantly increased at day compared with baseline ( fig. and table ). bal hnp - and cxcl /il- only were also significantly increased at day compared with week in asthmatic subjects ( fig. and table ). there was a trend to increased bal neutrophils at day compared with baseline in asthmatic subjects (p = . ). we also measured bal il- , but no significant differences were observed, neither within groups at the different time points nor between asthmatics and controls at any time point (data not shown). relationship between bal neutrophils, soluble mediators, virus load, and clinical parameters bal hnp - measured at baseline was negatively correlated with bal viral load (r = À . , p = . ) in asthmatics only (fig. a) . bal viral load was available in asthmatic subjects only. unfortunately, the other samples got lost during a liquid nitrogen thawing over christmas/new year and were not available for analysis. bal hnp - at baseline was correlated with bal cxcl /il- at baseline in asthmatics only (r = . , p = . ). bal hnp - was the only parameter to be positively correlated with relative bal neutrophil numbers at day post-infection (in all subjects; fig. b ). at week , bal hnp - was also correlated with bal cxcl /il- (r = . , p = . ) in all subjects. bal cxcl /il- and cxcl /gro-a levels at day post-infection were correlated with peak nasal lavage virus load (r = . , p = . , and r = . , p = . , respectively) in asthmatics. bal cxcl /il- at week was correlated with bal neutrophils (in% of all non-epithelial cells; r = . , p = . ) in all subjects. there were no correlations between clinical parameters (fev or pef) and hnp - or il- levels. elafin levels at day post-infection were inversely related to maximal falls in pef (r = À . , p = . ) in asthmatics (fig. c ). we have investigated the effect of rv infection on the expression of cxc chemokines and antimicrobial peptides in a human experimental model of rv-induced asthma exacerbation. we show, in accordance with turner et al. [ ] , that the neutrophil-attracting chemokine cxcl /il- is significantly increased in asthmatics compared with normal controls. bal neutrophils tended to be increased in asthmatics at day compared with normal controls and their number was related to hnp - levels. significantly higher levels of the antimicrobial peptide hnp - were released into the airways of asthmatic patients compared with normal controls during infection. respiratory infections are the main triggers of asthma exacerbations. respiratory viruses are the most frequent pathogens, and human rvs are most frequently detected [ , ] . it has been shown that during naturally occurring virus-induced asthma exacerbations, neutrophils are recruited into the airways as part of the immune defence [ ] . the influx of neutrophils correlates with symptoms and parameters of airways obstruction such as fev [ ] . accordingly, we observed in our experimental model higher values of bal neutrophils at day after intranasal experimental infection. however, these changes were moderate, probably due to the small number of patients and the rather mild severity of the induced asthma exacerbations. recruitment into this intensive and burdensome study was difficult resulting in small numbers of patients. moreover, also due to ethical constraints, experimental exacerbations had to be mild in character. the results presented are thus also consequences of these requirements. symptoms and reductions in fev were significantly greater in asthmatics compared with controls as previously reported [ ] . it has been shown before that days after experimental rv infection, the inflammatory response of the upper airways is increased which is associated with increased symptoms and airways obstruction in asthmatics [ ] . we report that the increase in neutrophils is associated with higher hnp - levels. this suggests that neutrophils could be the major source of hnp - . to our knowledge, the only other cell type for which hnp - expression has been shown is cd-cd cells in blood [ ] . hence, we do not expect that there are any other relevant cellular sources of hnp - than neutrophils in bal. in favour of this is also the fact that cxcl /il- was the only chemokine significantly increased at day in asthmatics. it has been shown that hnp - can induce cxcl /il- [ ] , which may explain to a certain degree the significantly higher levels observed at day [ ] . significantly higher levels of cxcl /il- and a trend for higher cxcl /gro-a in bal at day were related to high virus load measured in nasal lavage. this may be a result of increased induction of cxcl /il- and cxcl /gro-a in asthmatics by rvs. it has been shown in vitro that rv infection of human respiratory epithelial cell line significantly increases cxcl /il- [ ] . moreover, it has been demonstrated that the intramuscular injection of synthetic hnp induces the transcript expression of genes encoding both pro-inflammatory cytokines (il- beta and tnf-alpha) and the chemokine cxcl /il- . furthermore, hnp showed chemotactic capacity towards leucocytes [ ] . these findings suggest that rv infection induces cxcl /il- , which has chemotactic activity towards neutrophils, thereby increasing neutrophil numbers in the airway which release hnp - which has properties that will further enhance neutrophilia. however, it has to be acknowledged that it is possible that increased defensin expression could also be an epiphenomenon of neutrophil activation and that other mechanisms, such as release of reactive oxygen species or other pro-inflammatory mediators, may at least also contribute to drive an asthma exacerbation. all cxc chemokines investigated here are chemoattractant for neutrophils, the major effector cells during asthma exacerbation and viral airway infection. interestingly, they signal through a common receptor (cxcr ) [ ] . cxcr is required for rv induction of neutrophilic airway inflammation and development of airway hyperresponsiveness as recently demonstrated in a mouse model [ ] . hence, cxcr could be an interesting target for therapy in rv-induced asthma exacerbations [ ] . specific cxcr / receptor antagonists are already in clinical development [ ] . why might rv infection lead to increased expression of human neutrophil peptides? antimicrobial peptides such as hnp - are important effector molecules of neutrophils. it was suggested that a-defensins (hnp - ) cannot directly inactivate non-enveloped viruses such as rvs [ ] . however, recent research showed that this is not completely true. it was shown that human a-defensins (hd- ) can block adenovirus uncoating [ ] . moreover, it is known that hnp - are potent antagonists of infection by both cutaneous and mucosal papillomavirus types by blocking virion escape from endocytic vesicles [ ] . thus, hnp - do have direct antiviral properties against non-enveloped viruses and might also have direct antiviral properties against rv infection. in addition, hnp - might have indirect antiviral effects. they have recently been shown to inhibit hiv- replication even when added h postinfection [ ] . moreover, it was demonstrated that hnp can affect the ability of adenoviruses to infect epithelial cells [ ] . a-defensins have chemoattractant properties towards both cd + and cd + /cd ra + t cells [ ] . increased levels of a-defensins during viral infection could therefore recruit both cd + and cd + cells to the airways. this may enhance antiviral immunity as it has been shown in a variety of murine models that hnps enhance antigen-specific humoral and cellular immunity [ ] [ ] [ ] . however, one has to bear in mind as laid out above that increased defensin expression could also be an epiphenomenon of airways neutrophila which is considered to contribute to asthma exacerbations. elafin levels were significantly higher in asthmatics at day compared with normal controls and were times higher than those of hnp - . however, elafin levels were not significantly increased at day compared with baseline or convalescence. elafin levels at day post-infection were inversely related to maximal fig. . (a) the relationship between bronchoalveolar lavage (bal) hnp - levels and bal viral load at day post-infection was investigated in the two subject groups. in the asthmatic group (■), there was a significant inverse correlation between hnp - and bal viral load at day post-infection, which was not present in the normal group (□). (b) the relationship between bal hnp - levels and bal neutrophils at day post-infection was investigated in the two subject groups. in the asthmatic group (■), there was a significant correlation between hnp - and bal neutrophils at day post-infection. the same relationship was observed in the normal group (□). (c) the relationship between bal elafin levels at day post-infection and peak flow maximal fall was investigated in the two subject groups. in the asthmatic group (■), there was a significant inverse correlation between bal elafin and peak flow maximal fall at day post-infection, which was not present in the normal group (□). falls in pef. this correlation in the absence of a significant increase in response to rv infection (probably due to low subject numbers) must be interpreted with caution. it might suggest that insufficient expression of this molecule might lead to more pronounced functional consequences of rv infection in asthmatics. however, this has to be supported in further experimental and/or clinical studies. bal cxcl /nap- levels were significantly lower in asthmatics at day compared with controls. it has been shown in a ferret model using microarray analysis that infection with -h n a/california/ induced the expression of multiple chemokines including ccl / mcp- , ccl /mcp- , ccl /mcp- , ccl /elc, cxcl /nap- , and cxcl /ip- [ ] . a recent study found that increased ccl /rantes and cxcl /nap- expression was associated with neutrophil activation in severe stable copd. it seems that cxcl /nap- plays a role in the local innate immune response and that dysregulation of the expression of this molecule might result in neutrophil dysfunction [ ] . clearly, this hypothesis has to be investigated further. this study has strengths and weaknesses. the major strength of this study is the study design. experimental rv infection in humans provides an excellent model of virus-induced asthma under controlled conditions including application of a standard dose of a single virus serotype and standardized clinical data collection. invasive sampling can be carried out under controlled conditions repeatedly and at accurately defined time points. however, this elaborate study design is extremely labour-intensive which accounts for limitation of number of subjects that can be included in such a study. thus, subject numbers have to be small. for safety reasons, only mild asthmatics could be included into the study. this limits the ability to study more severe forms of asthma. another important aspect is that bal sampling time points had to be limited to due to the invasive character of this investigation, and it seems possible that the time points chosen (baseline, days and weeks after experimental infection) do not correspond to the peak changes in cxc chemokine and/ or amp expression. moreover, the analysis of soluble markers in respiratory secretions is complex because of dilution, modification, and degradation. nevertheless, we found significant differences between asthmatics and normal controls which results from meticulous patient characterization before inclusion. regarding in vitro findings and preliminary in vivo data, our results deliver direct evidence that rv infections increase levels of a-defensins in the airways. this has been assumed as rv infections lead to marked neutrophil infiltration and degranulation in the airways [ , ] . this finding is of possible importance as neutrophil degranulation is associated with clinical severity of virus-induced asthma [ ] . this is the first study showing increased expression of neutrophil antimicrobial peptides in a well-defined human model of experimental rhinoviral infection of asthmatics. we propose that rv infection in asthma leads to increased release of cxcl /il- thereby attracting neutrophils into the airways where they release hnp - which further enhances airway neutrophilia. further studies are warranted to better understand the role and importance of these cells and molecules in asthma exacerbations in order to identify possible new targets for therapy. author's contributions gr was involved in the hypothesis delineation, the analysis and interpretation of data, and wrote the manuscript. sdm was involved in the conception, hypotheses delineation, and design of the study, the analysis and interpretation of data and had substantial involvement in the revision of the manuscript prior to submission. jh, tk, hp, and vls were involved in the acquisition of the data and the analysis and interpretation of data. mrk, omk, las, pm, and mre were involved in the conception, hypotheses delineation, and design of the study, acquisition of the data and the analysis and substantially revised the manuscript prior to submission. slj was involved in the conception, hypotheses delineation, and design of the study, the analysis and interpretation of data and in writing and revising the article prior to submission. frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study respiratory viruses and exacerbations of asthma in adults community study of role of viral infections in exacerbations of asthma in - 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- journal: respir res doi: . /s - - - sha: doc_id: cord_uid: n xvwkf background: influenza a viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. application of high gm-csf levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. methods: mice were infected intranasally with influenza a virus (pr strain). supra-physiologic levels of gm-csf were induced in the airways using the double transgenic gm-csf (dtgm) or littermate control mice starting on days post-infection (dpi). assessment of respiratory mechanical parameters was performed using the flexivent rodent ventilator. rna sequence analysis was performed on facs-sorted airway macrophage subsets at dpi. results: supra-physiologic levels of gm-csf conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (bal) fluid to near baseline levels. transcriptome analysis, and subsequent validation elisa assays, revealed that excess gm-csf re-directs macrophages from an “m -like” to a more “m -like” activation state as revealed by alterations in the ratios of cxcl and ccl in bal fluid, respectively. ingenuity pathway analysis predicted that gm-csf surplus during iav infection elicits expression of anti-inflammatory mediators and moderates m macrophage pro-inflammatory signaling by type ii interferon (ifn-γ). conclusions: our data indicate that application of high levels of gm-csf in the lung after influenza a virus infection alters pathogenic “m -like” macrophage inflammation. these results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. each year, influenza a virus (iav) affects a significant proportion of the population [ ] and causes pathologic changes both through direct cellular toxicity causing desquamation, de-ciliation, and cell death, and through indirect effects by stimulating an anti-viral immune response leading to collateral injury [ ] . this combination can lead to an ards-like syndrome characterized by increased capillary leak, oxygen diffusion difficulty and ventilation/perfusion mismatch [ ] . immune strategies that protect the host's lung function while still allowing for an adequate immune response to clear the viral load and resolve virus-induced pneumonia are needed. a number of pre-clinical studies have tested prophylactic gm-csf both as vaccine adjuvant and local supplementation against iav infection with encouraging results [ ] [ ] [ ] [ ] . the effect of local elevation of gm-csf on iav infection in the lung has been investigated in transgenic models with expression of gm-csf under the control of constitutive or doxycycline-inducible promoters in lungs of alveolar or small airway epithelial cells of gm-csf knockout (csf −/− ) mice [ , ] . differential effects on morbidity and mortality from iav infection in these studies was associated with increased alveolar macrophage (am) numbers in the constitutive gm-csf expression models [ , ] and am differentiation in the gm-csf-inducible model [ ] . differential results on morbidity and survival were also obtained after prolonged or brief administration of supra-physiological levels of gm-csf before or at the onset of iav infection [ ] . the question of whether therapeutic administration of gm-csf to the airways after establishment of the infection would confer protection has never been addressed. in this study we use a more clinically relevant model to examine whether supra-physiologic levels of gm-csf in the airways, induced after iav infection at the peak of virus replication, provided therapeutic benefit. using gm-csf-inducible mice on the wt c bl/ genetic background we show that airway gm-csf over-expression starting at days post infection (dpi) provides protection from mortality and prevents the degeneration of multiple lung mechanical properties. to examine the mechanism of protection conferred by therapeutic gm-csf levels, we measured respiratory and biochemical parameters of lower airway disease, and analyzed the transcriptome of facs-sorted ams and exudative macrophages (em) from iav-infected mice. our findings demonstrate that gm-csf restores proteostasis of exudate proteins and redirects responsiveness of ams and ems from an m -like to an m -like activation state, and prevents mortality from influenza-induced ards. the double transgenic gm-csf (dtgm) mice were bred as previously described [ ] , but this time on the wild-type c bl/ j background. littermate control (lm) mice were defined as being single transgenic littermates of dtgm mice that were only positive for the scgb a -rta and thereby did not have the cmv-gm-csf gene, which may potentially be virally induced in the absence of tetracycline (doxycycline) [ ] . dtgm mice and lm controls were exposed to mg/ml doxycycline in drinking water, and doxycycline-containing drinking water was replenished every - days. both male and female mice were used for all experiments; all mice were sex-and age-matched to control mice. the influenza strain a/puerto rico/ / (pr ) virus was a kind gift of dr. kevan hartshorn, and was grown in the chorio-allantoic fluid of ten ( ) day old specific pathogen free avian supplies (spafas) chicken eggs purchased from charles river laboratories (wilmington, ma) and purified on a discontinuous sucrose gradient as previously described [ ] . mice were anesthetized with ketamine/xylazine and intranasally (i.n.) infected with iav virus in μl of pbs. mice were infected in a bsl biosafety cabinet and housed within filter-top microisolator cages in the pulmonary immunology and physiology (pip) core, a bsl facility in the department of comparative medicine's animal facility at penn state university college of medicine. mice were observed at least twice daily during infections to assess morbidity and mortality. based on our experience at our facility in the last years and the variable clinical presentation of the influenza infection, we used other metrics to monitor morbidity in addition to mouse body weight curves [ ] . mice that exhibited immobility, ruffled hair, and labored breathing that had no chance of recovery, coinciding with approximately % of body weight loss, were euthanized by ketamine/xylazine overdose and cervical dislocation, and counted as dead. alternatively, mice that were sleeping but had normal breathing and body appearance, i.e., no ruffled hair or labored breathing, reached up to - % body weight loss and then began to recover normally. mice with favorable prognosis but with % body weight loss or greater were provided supportive care with food and hydrated gel packs at the bottom of the cage. we have not found a pattern of clinical disease specific to mouse genotype or gender in untreated mice. mice were anesthetized with ketamine/xylazine ( mg/kg and mg/kg, i.p., respectively). the trachea was cannulated via tracheostomy with a g blunt needle and the cannula was secured in place with a suture. mice were kept sedated using isoflurane inhalation (maintenance dosing, - % of inspired air) through the flexivent and were paralyzed with mg/kg vecuronium i.p. mice were ventilated using baseline settings of positive end expiratory pressure (peep) cm h o, tidal volumes (vt) ml/kg, respiratory rate (rr) breaths per minute (bpm) and an fraction of inspired oxygen (fio ) of . . oxygen saturations were measured using the mouseox plus pulse oximeter (starr life sciences, oakmont, pa, usa) via the thigh sensor. lung mechanic parameters were generated from the flexivent rodent ventilator using the forced oscillation technique as previously described [ ] . bronchoalveolar lavage (bal) protein measurements bal samples were collected as previously described, and after centrifugation at g for min, bal supernatants were removed and immediately frozen at − °c until batch analysis. proteins were measured with kits as detailed in additional file : table s . elisa plate absorbance was measured at nm with a spectramax m uv/vis/fluorescence - plate reader (molecular devices, sunnyvale, ca). cytokines were measured by elisa as described above or by procartaplex cytokine & chemokine -plex mouse panel a (thermo fisher scientific) via luminex magpix multiplex array (luminex). after iav infection, the entire lung was removed from each mouse and placed in ml of trizol (thermo fisher scientific, waltham ma), weighed, homogenized on ice using a polytron homogenizer for - s intervals, and frozen in aliquots at - °c until rna extraction. dna was then extracted using chloroform and rna was precipitated using isopropanol. quantitative rt-pcr for iav m copies per lung was performed as previously described [ ] using the following primers: influenza a/ /puerto rico/ m gene sense: '-aagaccaatcctgtcacctctga- ′ and antisense: ' caaagcgtct-acgctgcagtc - ′ primers, and the mediator probe sequence: ′-/ fam/ tttgtgttcacgctc-accgt/ -tamsp/ - ′. data are expressed as m viral copies per lung. single cell suspensions were prepared from bal and lung as described in supplemental methods. single cell suspensions from lung digests were placed at °c and then surface stained in hank's buffered saline solution (hbss) with % fbs with fluorochrome-conjugated monoclonal antibodies (additional file : table s ), and then stained with a fixable viability dye. for facs-sorting, bal cells were recovered and placed at °c and then surface stained in hank's buffered saline solution (hbss) with % fbs with fluorochrome-conjugated monoclonal antibodies (additional file : table s ) and -aminoactinomycin d ( -aad) was used to assess viability just prior to acquisition. all flow cytometric data were collected in the penn state hershey flow cytometry core facility using an lsr ii (becton dickinson, bd) instrument, and all facssorting was performed using a facsaria (bd) high speed cell sorter. cells were sorted directly into rna-bee to isolate rna for further rna-sequencing (rna-seq). all facs data analysis was performed using flowjo version . (treestar, mountain view, ca). rna was phase separated using chloroform and the aqueous phase containing rna was removed following centrifugation and precipitated overnight at − °c using ice cold isopropanol. rna was washed with % ethanol then solubilized in rnase-free water. optical density values of extracted rna were measured using nanodrop (thermo fisher scientific) to confirm an a :a ratio above . . rna integrity number (rin) was measured using bioanalyzer (agilent) rna pico kit to confirm rin above . the cdna libraries were prepared using the smarter® ultra® low input rna kit for sequencing -v (clontech) followed by nextera xt dna library prep kit (illumina) as per the manufacturer's instructions. the unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. the final product was assessed for its size distribution and concentration using bioanalyzer high sensitivity dna kit (agilent technologies) and kapa library quantification kit (kapa biosystems). the libraries were diluted to nm in eb buffer (qiagen) and then denatured using the illumina protocol. the denatured libraries were diluted to pm by pre-chilled hybridization buffer and loaded onto truseq sr v flow cells on an illumina hiseq (illumina) and run for cycles using a single-read recipe (truseq sbs kit v , illumina) according to the manufacturer's instructions. illumina casava pipeline (released version . , illumina) was used to obtain de-multiplexed sequencing reads (fastq files) passed the default purify filter. additional quality filtering used fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit) to keep only reads that have at least % of bases with a quality score of or more (conducted by fastq_quality_filter function) and reads left with bases or longer after being endtrimmed with reads with a base quality score of b (conducted by fastq_quality_trimmer function). a bowtie index was built for the mouse reference genome (grcm ) using bowtie version . . . the rna-seq reads of each of the samples were mapped using tophat version . . [ ] supplied by ensembl annotation file; grcm . .gtf. gene expression values were computed using fragments per kilobase per million mapped reads (fpkm). differential gene expression was determined using cuffdiff tool which is available in cufflinks version . . [ ] supplied by grcm . .gtf. normalization was performed via the median of the geometric means of fragment counts across all libraries, as described in anders and huber [ ] . statistical significance was assessed using a false discovery rate threshold of . . we arbitrarily chose to further analyze the % most highly expressed gene transcripts in am or em cell populations from iav-infected dtgm or lm mice using ingenuity pathway analysis (ipa, www.qiagen.com/ingenuity) to identify upstream signaling pathways. significance was measured by fisher's exact test with a q < . cut-off. all statistical analysis was performed using jmp . . software (sas, cary, nc). normally distributed data were analyzed using student's t-test, and non-normally distributed data using wilcoxon signed-rank test. survival analysis was calculated by using the log-rank test. all data points are means ± standard error of the mean (sem) unless otherwise stated. graphs were created using prism for mac os x (graphpad, la jolla, ca). to characterize the pathogenicity of our h n pr iav preparation virus, wild-type c bl/ j mice (the jackson laboratory, ma) were purchased and we determined the lethal dose % (ld ) of our pr iav preparation. female wild-type mice were much more susceptible than males with an ld approximately fold lower than males ( vs. ffu, female and males respectively, additional file : figure s a -d). airway gm-csf levels were conditionally increased following iav infection using a doxycycline inducible promoter in the dtgm mouse model, formerly named the tet-gm +/+ , as previously described [ ] . in this conditional transgenic mouse model gm-csf is expressed and secreted by airway club cells via the club cell (cc , scgb a ) promoter after oral administration of doxycycline ( mg/ml in water ad libitum) (fig. a) . importantly, in the absence of infection, bal fluid levels of gm-csf in dtgm mice are near the limit of detection, similar to littermate controls (additional file : figure s a ), and their alveolar macrophages appear identical by multiparameter flow cytometry. once doxycycline is administered, bal levels of gm-csf peak after approximately h reaching levels of approximately pg/ml in . ml of recovered bal fluid and in preliminary experiments the dtgm mice were either administered or not administered doxycycline to create a condition of elevated vs. wild-type levels of airway gm-csf, respectively [ ] . however, these preliminary experiments demonstrated that low levels of gm-csf from the scgb a promoter in dtgm mice were endogenously induced by interferons during iav infection (additional file : figure s a ), a finding that has previously been reported [ ] . therefore, all subsequent experiments compared the dtgm to lm groups, both exposed to doxycycline, to examine the effect of elevated (dtgm) as opposed to wild-type (lm) levels of airway gm-csf, while also controlling for any off-targets effects of doxycycline. to address our research question of whether "treatment" with gm-csf during severe iav infection would improve survival, dtgm and lm mice were infected i.n. with approximately ld (differential dosing based on sex) of pr iav and were administered doxycycline in drinking water. gm-csf overexpression (dtgm) conferred a significant survival advantage as compared to wild-type levels (lm, fig. b , **p < . ). weight loss and recovery were similar in the two groups, however, because of survivor bias likely artificially elevating the average weights of surviving lm mice (fig. c) . of note, doxycycline treatment of lm mice had no effect on survival whereas doxycycline-untreated dtgm mice demonstrated a survival advantage over untreated lm mice, suggesting that even low levels of gm-csf can confer some survival benefit (additional file : figure s b ). lower respiratory tract iav infection can lead to impaired oxygenation due to v:q mismatch and decrease lung compliance due to the infiltration of inflammatory cells and an increase in lung water weight [ ] . given the ability of gm-csf to confer survival, we expected elevated gm-csf levels to improve arterial oxygen saturations (% spo ). however, gm-csf did not significantly increase median oxygen saturations (% spo ) levels as compared to lm mice at either or dpi (data not shown). to gain insight into whether gm-csf conferred any lung mechanical benefits, lung mechanics scans were performed by the forced oscillation technique and pv loop curves were generated (fig. a, b) . as expected, the pv curve flattens with iav infection due to decreased static compliance (cst), but we were surprised that compliance continued to fall from days to ( fig. a-c) . while gm-csf did not affect cst (fig. c) or total system resistance (rrs, fig. d ), dtgm mice demonstrated less tissue damping or peripheral airway resistance (g, cmh o/ml, fig. e) , and significant preservation of newtonian or central airway resistance (rn, cmh o*s/ml, fig. f ) and curvature of the deflation limb of the pv curve, a measure of maintenance of alveoli and small airway recruitment (k, /cmh o, fig. g ) at dpi. given that two of the lung mechanical parameters that are maintained by gm-csf, k and g, are correlated with dynamic processes at the small airway or alveolar level, namely alveolar size [ ] and changes in tissue physical properties of small airways [ ] respectively, and which can change with lung interstitial edema [ ] , we hypothesized that gm-csf may improve lung capillary barrier function and/or enhance alveolar fluid clearance. as surrogate of alveolar fluid content, we measured the concentration of total protein in bal fluid and found that gm-csf overexpression decreased bal fluid total protein levels at (peak inflammation) and dpi (early resolution phase) (fig. a) . to further investigate this difference in bal fluid protein content, we examined the concentration of various serum and lung-specific proteins including mouse serum albumin ( kda), as well as two larger proteins, alpha- -macroglobulin ( kda monomer, kda tetramer) and immunoglobulin m (igm, kda monomer, kda pentamer) as markers of capillary leak [ ] . gm-csf significantly decreased alpha- macroglobulin levels at dpi (fig. b) , but did not significantly decrease other markers of capillary leak including albumin or igm (additional file : figure s a-b) . we also directly assayed the lung epithelial barrier function with fitc-labeled dextran (mw , ), but surprisingly no differences in epithelial barrier function could be detected at dpi (data not shown). additionally, we also investigated whether gm-csf overexpression during iav increased bal levels of the epidermal growth factor family member, amphiregulin. gm-csf overexpression in uninfected mice elevated levels of amphiregulin, though iav infection also induced amphiregulin and gm-csf did not further increase these levels ( fig. c) . lastly, we assessed whether elevated gm-csf levels during active infection affected viral clearance. at dpi, the peak of virus levels in our model, we recovered - × m -copies total lung copies of iav pr matrix (m ) via rt-pcr and the viral copies decreased to . - × by dpi, though there was no statistically significant difference with gm-csf overexpression (fig. d) . alveolar macrophages have been shown to be necessary for protection from iav [ ] [ ] [ ] [ ] [ ] [ ] [ ] . gm-csf is known to mediate the proliferation and differentiation of monocytes and macrophages; studies using constitutive expression or gm-csf administration before iav infection models both documented an increase in am numbers [ , , ] . therefore we hypothesized that gm-csf would protect siglecf + ams from viral-induced depletion and would increase numbers of total airway (bal-recovered) macrophages. to investigate this immune cells were characterized and enumerated in single cell suspensions of bal and lung enzymatic digests by multi-parameter flow cytometry using a -color panel of macrophage and granulocyte surface markers (fig. a) . we specifically focused on the two predominant airway macrophage populations present during active iav infection: f / + , cd b neg/dim , siglecf + cells to discriminate alveolar macrophages (ams) and f / + , cd b + , siglecf neg/dim cells that have been termed exudative macrophages (ems) [ ] . our typical yield of ams recovered from bal fluid of an uninfected mouse is approximately , cells. at dpi, at the height of the inflammatory response to iav, the number of ams to simulate a therapeutic model of gm-csf administration doxycycline was administered to both dtgm and lm control mice starting days after i.n. infection with pr iav. doxycycline-containing water was protected from light and changed every three days (a). dtgm (n = , red circles/lines) and lm control (n = , black squares/lines) mice were administered approximately ld of iav pr virus i.n. and administered doxycycline in water starting on + dpi, and the effects on survival and body weight are shown. mice were euthanized if they lost > % body weight and were moribund. gm-csf over-expression (dtgm mice) conferred a significant survival benefit (b) but not a significant effect on weight loss/recovery (c) as compared to wild-type levels (lm mice). results shown represent three independent experiments (**p < . ) recovered was much lower, and gm-csf overexpression did not serve to increase this number (fig. b) . in contrast, ems become the predominant airway macrophage during iav infection at this time point, but again, gm-csf overexpression did not affect em cell numbers (fig. b) . while gm-csf overexpression did not change the number of macrophages, we hypothesized that it changed their phenotype. this is not a new concept as the primary function of gm-csf on ams is to induce differentiation and activation [ , , ] . despite attempting to discriminate the macrophage populations by multiple cell surface markers, we could not distinguish the iav-responding macrophages further than alveolar and exudative macrophages as described in fig. a . therefore, we sought to determine whether gm-csf affected the transcriptomes of the am and em populations independently by first facs-sorting the airway macrophages. facs-sorted airway macrophages from bal fluid were obtained at dpi, the time point where the survival curves of the dtgm and lm mice begin to diverge, and next generation rnasequencing was performed on the sorted am and em populations. using an unbiased approach, we identified the transcripts that were significantly affected by gm-csf over-expression during iav infection by comparing the mean value of each transcript from the dtgm and lm groups. for direct comparisons, transcripts that had a mean value of zero ( ) fkpm in one of the groups were not analyzed. of the , genes available in the reference genome, in the am population transcripts were significantly different between the groups with gm-csf over-expression leading to up-regulation of the chemokines ccl , cxcl , and ccl , and the down-regulation of cxcl , and arg , the prototypic marker of m macrophage polarization (fig. a) [ ] . in comparison to ams, in ems gm-csf induced more transcripts than it inhibited. only fig. flow cytometric discrimination of alveolar and exudative macrophages by surface marker expression. representative facs plots from an iav-infected lm mouse at dpi, which detail our -color flow cytometry gating strategy of single cell suspensions from bal and enzyme-digested lung (a). alveolar macrophages (am) were designated as f / + siglecf + cd b neg/dim , whereas exudative macrophages (em) were designated as f / + siglecf neg/dim cd b+. supra-physiologic gm-csf levels during iav infection had no effect on the absolute number of either airway (bal-recovered) am or em cell numbers at dpi (b) six transcripts were down regulated by gm-csf including lipg, cxcl and ccl , while gm-csf overexpression induced multiple transcripts in ems including dcstamp, retnla, irgc , mmp , and ccl (fig. b) . our unbiased analysis demonstrated that gm-csf overexpression during iav led to the up-regulation of some transcripts associated with m macrophages including matrix metalloprotease , mmp , and ccl , and the down-regulation of some m macrophage-associated transcripts such as cxcl and cxcl . therefore we examined the effect of gm-csf on multiple canonical and novel macrophage polarization markers [ ] . interestingly, while gm-csf tended to down-regulate m transcripts and up-regulate m transcripts, this effect was not absolute in either ams or ems (fig. c-f ). to validate these macrophage transcript differences we measured the chemokines ccl and cxcl , and the m -associated metalloprotease, mmp , in bal fluid by elisa. ccl was significantly induced by gm-csf (not only during iav infection, but also when gm-csf was induced in the absence of iav (fig. a) . in comparison, negligible amounts of cxcl were present in uninfected mice regardless of gm-csf induction, whereas with iav fig. characterization of the changes in transcriptome patterns of airway macrophages during iav infection. bal airway macrophages were sorted using the gating strategy described in fig. a and next generation rna-sequencing was used to profile the complete transcriptome data of ams (a, orange bars) and ems (b, blue bars) at dpi, the time point at which the survival curves diverge (n = mice per group). the effect of supraphysiologic gm-csf levels on each of the , sequenced macrophage genes was examined: differential gene expression was determined using with transcripts having a q-value < . being included. the relative expression of each transcript was calculated using the equation, log expression ratio (dtgm:lm) = log ðx transcript dtgm ) -log (x transcript lm ), and the differential expression of transcripts is shown. to investigate the impact of gm-csf on m /m macrophage polarization, the log expression ratios were plotted against known m and m macrophage-associated transcripts from ams (c, d) and ems (e, f) infection gm-csf overexpression there was a trend toward decreased expression (fig. b, p = . ). the concentration of mmp was approximately -fold higher in gm-csf overexpressing mice at dpi as compared to lms (fig. c, p < . ) . we also examined the ratio of the two chemokines (cxcl : ccl ) as an intrinsic property of the balf to probe macrophage polarization by chemokine expression and supra-physiologic gm-csf levels significantly decreased this ratio more than ten-fold in iav-infected mice (fig. d , p < . ). lastly, we attempted to determine which signaling pathways were affected by gm-csf overexpression during iav infection by analyzing our transcriptomes with ingenuity pathway analysis (ipa) software (qiagen). we analyzed the effect of gm-csf overexpression on the mean log expression ratios for the % most expressed genes in each of the macrophage type groups (am vs. em). the ipa software allows the construction of an upstream analysis that calculates the likelihood that an upstream regulator is involved given the gene set provided (p-value of overlap), as well as a composite score of activation depending on the state of downstream genes being increased or decreased in quantity (activation z-score). for both the upstream regulator analysis, and the subsequent canonical pathway analysis, we used a stringent p-value of overlap cutoff of e- . ipa predicted that gm-csf activates (table a ) several upstream regulators of signaling pathways in both ams and ems including il- receptor alpha (il ra), transcription factor tripartite motif-containing (trim ), and the atypical chemokine receptor (ackr ). conversely, ipa predicted that gm-csf over-expression inhibited multiple inflammatory signaling pathways in both ams and ems including interferon regulatory factor (irf ), irf , interferon gamma (ifng), interferon alpha/beta receptor (ifnar), tir domain-containing adapter molecule (ticam , or trif), signal transducer and activator of transcription (stat ), rapamycin-insensitive companion of mammalian target of rapamycin (rictor), toll-like receptor (tlr ), dexd/hbox helicase (ddx , or retinoic acid-inducible gene [rig- ]), and inhibitor of nuclear factor kappa-b kinase subunit beta (ikbkb). in terms of canonical pathway analysis one pathway, "eukaryotic initiation factor (eif ) signaling", was activated in both ams and ems, whereas "fc-γ receptormediated phagocytosis in macrophages and monocytes" was inhibited in both populations (table c, d) . "interferon signaling" was inhibited in ems (table d) , and trended towards significance in the am population [−log(p-value) . , z-score − . ], even though the levels of type i, ii and iii interferons were unchanged in fig. effect of gm-csf overexpression on airway levels of ccl , cxcl and mmp . mouse ccl (a), cxcl # (b), and mmp # (c) were measured by elisa in bal fluid from doxycycline-treated lm (black) and dtgm (red) uninfected and iav-infected ( dpi) mice. furthermore, the ratio of cxcl :ccl # in each bal sample was determined to examine the relative effect of supraphysiologic gm-csf levels on macrophage chemokine polarization (d). results from three independent experiments. ( # please note the log scale, *p < . , **p < . ) bal fluid from dtgm as compared to wt mice (additional file : figures s a-c ). in this study we examined the effect of elevated gm-csf levels during iav infection on clinical, lung physiologic and biochemical markers in a mouse model, and then used rna-sequencing to ascertain the differential effects of elevated gm-csf levels on the transcriptomes of the two predominant airway macrophages present during the peak of iav infection. our finding that elevation of airway gm-csf during active iav infection confers protection from mortality from iav is novel. multiple preclinical mouse studies have described the observations that the absence of gm-csf increases susceptibility to iav [ , , ] , while supra-physiologic levels of gm-csf achieved by constitutive overexpression or exogenous administration are beneficial [ , , ] . importantly, however, the publications that have demonstrated positive effects of supra-physiologic levels of gm-csf against iav infection have used either constitutive expression models [ ] [ ] [ ] or have administered gm-csf either before [ , ] or on the day of infection [ ] . to our knowledge this is the first description of the use of a therapeutic model of gm-csf wherein it is "administered" to the airways well after establishment of the infection (+ dpi) and still confers protection. table ingenuity pathway analysis predictions of the effects of supra-physiologic levels of gm-csf on airway macrophages during iav. bal airway macrophages were sorted and rna-sequencing was performed to compare the gene expression between iav-infected lm (n = mice) and dtgm (n = mice) treated with doxycycline at dpi. using the means of each group, the % ( genes) most expressed transcripts from each of the genotypes, dtgm and lm, were analyzed using qiagen's ingenuity pathway analysis (ipa) software. ipa was used to identify differential upstream regulators between ams (a) and ems (b) of dtgm and lm mice, and upstream regulators were included in the table if their p-value of overlap was < e- and the activation z-score was < − or > + . ipa was also used to identify differential effects of gm-csf on canonical pathways of ams (c) and ems (d). ingenuity canonical pathways were included in the table if their -log(p-value) was > and the z-score of pathway activation was < − or > + gm-csf over-expression led to an increase in macrophage expression and bal fluid levels of ccl and mmp , whereas a decrease in cxcl or monokine induced by gamma interferon (mig). these protein data, in addition to our macrophage transcriptome data, suggest that high levels of gm-csf push the typically classically activated m -like monocytes/macrophages in the lung during iav towards an m -like phenotype. interestingly, a recent investigation showed that the presence of m -like monocytes are a major determinant of iav pathogenicity in patients and strengthened this notion with a mouse model demonstrating that adoptive transfer of m as opposed to m macrophages results in better outcomes [ ] . the observation that gm-csf is pushing macrophages towards an m -phenotype is in stark contrast to a large body of in vitro literature that defines m monocytes/macrophages as being induced by gm-csf, whereas m monocytes/macrophages are differentiated by macrophage colony-stimulating factor (m-csf) [ ] [ ] [ ] . on the other hand, alveolar macrophages from gm-csf-deficient csf −/− mice exhibit a mixed m /m phenotype, not a strictly m phenotype as in vitro data would suggest [ ] . and our data also suggests that the polarization was not at all absolute: e.g., in ams, gm-csf led to lower transcript levels of the prototypic m macrophage marker, arg (fig. a) . thus, while the m /m macrophage polarization schema has been helpful [ , ] , perhaps a more nuanced view of macrophage polarization [ ] , where their intrinsic differentiation plasticity allows them to attend to specific needs of their local immune environment [ ] , could explain these results. ipa also predicted the activation of the il- receptor alpha-chain in both ams and ems. given that il- levels in bal fluid were not elevated in dtgm as compared wt mice (additional file : figure s d ), it is possible that gm-csf overexpressing during iav somehow potentiates il- signaling in the lung microenvironment. the role of interferons during iav infection is also nuanced. while it has been shown using ifnar −/− and ifngr −/− mice that interferon signaling is necessary for protection from iav [ ] , it is possible that this requirement only extends to epithelial cells. interferon-γ may not be necessary during iav infection and may in fact be detrimental, e.g., nitrogen oxide synthase deficient (nos −/−) mice are more protected from iav [ ] , and sun and metzger demonstrated that treatment with an anti-ifnγ mab clone xmg . had little effect on the course of the viral infection, but inactivation of ifn-γ protected against secondary bacterial pneumonia [ ] . recently, califano et al. showed that ifnγ −/− mice on both the balb/c and c bl/ backgrounds demonstrated improved survival to lethal iav infection [ ] . in their model, ifnγ serves to restrict protective innate lymphoid cell group (ilc ) function, whose production of il- and amphiregulin may improve lung barrier function. another group has also demonstrated that gm-csf can induce amphiregulin in a smoke model of copd followed by iav infection [ ] , however our data (fig. c) suggest that pretreatment with gm-csf is necessary for this effect on amphiregulin levels. furthermore, our gm-csf over-expression is started after iav infection, amphiregulin levels at dpi were not different in gm-csf over-expressing mice, and therefore amphiregulin is likely not an active player in our model. it is possible that our inducible gm-csf model may be replenishing gm-csf that otherwise would be produced by ilc s whose functions have been restricted by ifnγ [ ] . our data suggest that high levels of gm-csf inhibit interferon signaling in airway macrophages, though the mechanism is not clear. canonically, gm-csf signaling acts through jak /stat [ ] , though the beta-chain itself can activate nf-kb, and this activation is dependent on tnfr-associated factor (traf ) [ , ] , an e ubiquitin ligase with multiple immune functions [ ] . interestingly, our upstream analysis predicts that gm-csf activates trim , (aka tif α), a negative regulator of interferon signaling that acts by binding the retinoic acidresponsive element of the stat promoter [ ] , thus inactivating multiple interferon pathways. trim is also an e -ubiquitin ligase, and the tumor suppressor protein, p , serves as a ligand for both ligases: trim targets p for degradation [ ] while traf restricts p mitochondrial translocation [ ] . furthermore, a recent microarray study examining the relative pathogenicity of a mouse adapted strain of iav (ma-ca/ ) described negative inhibition of trim and early sustained interferon responses as important factors [ ] . however, we detected only very low levels of trim transcripts in our sorted airway macrophages, but gm-csf over-expression did lead to increased expression of another trim family member, trim , that also acts as a e ubiquitin ligase that can heterodimerize with other trims [ ] . future studies are needed to determine the exact cellular signaling pathways linking gm-csf and interferon. gm-csf enhanced exudative macrophage expression, and dpi bal fluid levels of mmp , or macrophage elastase, which is best known for its requirement for the development of smoke-induced emphysema in mice [ ] . however, it may also regulate acute inflammatory responses by proteolysis of chemokines [ ] , and through its divergent effects on ifn-α signaling depending on its intracellular (activating) vs. extracellular (inactivating) localization [ ] . a recent report demonstrated in two separate mouse models inflammation (peritonitis and arthritis) that macrophages resolve inflammation through multiple mechanisms via mmp including dampening neutrophil infiltration, clearing actin and fibrin from nets, terminating complement activation, and by activating prothrombin thus exhibiting procoagulant activity [ ] . cd + t cells and stat / , at least in a mouse model of pneumocystis pneumonia, are necessary for m macrophage mmp expression and relm-α and ccl production [ ] . while our data suggest that gm-csf may block m -like polarization in the lung during iav infection, it is not yet clear what in the lung microenvironment could promote m -like macrophage responses. recently it was shown that macrophage polarization may be pushed towards a il- dependent pathway in the lung and liver by the presence of surfactant protein a (sp-a) and complement component c q, respectively [ ] . the relationship between supraphysiologic gm-csf levels and sp-a during iav infection remains to be investigated and will be the subject of future studies. our current model of gm-csf induction on the wildtype background differs from our previous work using the inducible model generated on the gm-csf knockout (csf −/− ) genetic background [ ] . the present study is not confounded by prior immaturity of ams and defective surfactant catabolism, nor potential defects in migratory dendritic cell subsets, nk cells, and other myeloid cells outside the alveolar compartment in the lung and in other tissues of csf −/− mice [ ] [ ] [ ] [ ] , or disruption of gm-csf secretion by immune and non-immune cells that may elaborate gm-csf in response to infection. studies in csf −/− /spc-gm mice, in which t aecs express high levels of gm-csf constitutively, came to disparate conclusions as to the role of ams, dendritic cells and epithelial cells [ , ] in host resistance to iav infection. however, the life-long overexpression of gm-csf in the spc-gm +/+ model results in non-physiological proliferation of both t aec cells and ams [ ] that obscures assessment of temporal responses to iav. the spc-gm +/+ model also illustrates that prolonged lung exposure to supraphysiologic levels of gm-csf leads to desquamative interstitial pneumonia (dip) [ ] . we did not observe any similar findings of dip in our model, but this is not surprising given our model creates only a temporary doxycyclineinduced overxpression, and the overriding inflammatory effects of iav infection likely masks any differences. in our model, the ability of supra-physiologic levels of gm-csf to beneficially alter disease progression after iav infection delineates a time frame for possible future therapeutic intervention to arrest development of acute lung injury. in this regard, administration of gm-csf in humans has shown promise in the treatment of ards [ ] . concentration-dependent signaling via the gm-csf receptor affecting differentiation, proliferation, activation, and function of different effector cells has been studied extensively [ ] [ ] [ ] [ ] . lung function and organ 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molecular control and implications for immune homeostasis and therapy specific contributions of csf- and gm-csf to the dynamics of the mononuclear phagocyte system guide for the care and use of laboratory animals. national research council (us) committee for the update of the guide for the care and use of laboratory animals we would like to thank nate sheaffer and joseph bednarzyk from the penn state hershey flow cytometry core facility, as well as the institute for personalized medicine (ipm) at penn state hershey college of medicine, for assistance. we would also especially like to thank kevan hartshorn and mitchell white for providing the iav pr virus preparation used in all experiments. availability of data and materials all rna-seq data is available from the gene expression omnibus (geo) database, and the other datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. our data demonstrate that in vivo high airway levels of gm-csf profoundly rescue mice from lethal influenza pneumonia. while in vitro gm-csf is canonically described as an m -polarizing cytokine, our data demonstrates that in vivo, during iav infection, gm-csf instead temporizes the type ii interferon-induced m polarization of airway macrophages. the exact mechanism through which high levels of gm-csf block m macrophage polarization is still not known, and is the focus of our ongoing research. additional file : table s . all protein concentration measurements were made as described in the manuscript text using the reagents and kits listed. (tiff kb) additional file : table s . multi-parameter flow cytometry was utilized to characterize the alveolar and exudative macrophages as shown in fig the mouse influenza a virus infections and tissue harvesting were carried out by wg, md, tu, ly, sh and ekh. the rna sequence analyses were performed by yik, ps and jh. overall experimental design, analysis and interpretation were performed by esh with the mentorship of zcc. all authors read and approved the final manuscript.ethics approval and consent to participate all animal procedures were approved by the institutional animal care and use committee (iacuc) at pennsylvania state university college of medicine under protocols # and , , and were cared for as previously described [ ] . the regulation of the use of mice in research falls under the public health service policy on humane care and use of laboratory animals (phs policy), and is enforced by the office of laboratory animal welfare (olaw) under assurance number a - . in order to comply with the phs policy, our institution adheres to the us government principles for the utilization and care of vertebrate animals used in testing, research and training and the guide for the care and use of laboratory animals th edition [ ] . not applicable, the authors agree to pay the journal processing fee should the manuscript be accepted for publication. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -a nbmx l authors: stettler, gregory r.; moore, ernest e.; nunns, geoffrey r.; moore, hunter b.; huebner, benjamin r.; silliman, christopher c.; banerjee, anirban; sauaia, angela title: do not drink and lyse: alcohol intoxication increases fibrinolysis shutdown in injured patients date: - - journal: eur j trauma emerg surg doi: . /s - - -x sha: doc_id: cord_uid: a nbmx l introduction: high alcohol consumption has been associated with decreased fibrinolysis and enhanced thrombosis risk in cardiovascular disease. in trauma, alcohol has been associated with poor clot formation; however, its effect on fibrinolysis has not been fully investigated. we assessed the association of blood alcohol levels and fibrinolysis in trauma activation patients. methods: we queried our prospective registry of trauma activations from to . associations between viscoelastic measurements [rapid thrombelastography (rteg)] and blood alcohol level (bal) were determined and adjusted for confounders by a multinomial logistic regression. lysis phenotypes were defined by the % lysis in min (ly ) as follows: hyperfibrinolysis ≥ %, physiologic . – . %, and fibrinolysis shutdown < . %. results: overall, ( . %) had bal measured. there were ( %) patients that had no detectable bal, ( . %) had bal of – mg/dl, and ( . %) patients had bal > mg/dl. bal had a moderate, but significant inverse correlation with ly (rho = − . , p < . ), while there were no significant correlations between bal and other teg values. the distribution of fibrinolysis phenotypes varied significantly by bal levels (p < . , with high bal having more shutdown and less hyperfibrinolysis than the other two bal level groups. multinomial logistic regression showed that after adjustment for confounders, bal levels > mg/dl were independently associated with a threefold increase in the odds of shutdown compared to undetectable bal (or . , % ci . – . , p = . ). high bal was also significantly associated with higher odds of shutdown compared to low bal (or . , % ci . – . ). compared to physiologic fibrinolysis, fibrinolysis shutdown was associated with increased mortality (or . , % ci . – . ) and vfd < (or . , % ci . – . ). conclusion: in the injured patient, high blood alcohol levels are associated with increased incidence of fibrinolysis shutdown. this finding has implications for postinjury hemostatic resuscitation as these patients may be harmed by anti-fibrinolytics. further research is needed to assess whether the association with fibrinolysis is modified by the chronicity and type of alcohol consumed and whether anti-fibrinolytic therapy in intoxicated patients produces adverse effects. alcohol intoxication leads to metabolic and physiologic derangements that complicate the care of intoxicated trauma patients compared to their non-intoxicated counterparts [ ] . these physiologic changes include impaired cardiovascular function, blunting of catecholamine release leading to inadequate oxygen delivery to tissue and metabolic uncoupling, modulation of the innate immune system, and alterations in blood coagulation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . acute and chronic alcohol consumption can have varying effects, and distinct, sometimes opposite, effects on coagulation following alcohol intoxication have been described in the literature. some have described hypocoagulability in the intoxicated patient as evidenced by prolonged initiation of clot formation and decreased dynamics of clot formation resulting in a reduced risk of venous thrombotic events (vte) in trauma [ , ] . conversely, others have established a link between alcohol intoxication and increased risk of thrombotic complications through elevated levels of plasminogen activator inhibitor- (pai- ), the primary inhibitor of fibrinolysis [ , ] . this differential risk of adverse effects appears to be related to the amount of and timing within which ethanol is consumed [ , ] . while there is a decrease in fibrinogen and platelets reported with excess alcohol consumption [ ] , there is also a reported increase in coagulation factor vii, factor viii, and pai- in the acutely intoxicated [ , , ] . on the other hand, factor vii has been shown to decrease in chronic alcohol use, likely from liver disease and synthetic dysfunction [ ] . furthermore, there is a greater increase in pai- release compared to tissue plasminogen activator (tpa) in those who consume a larger amount of alcohol, thereby increasing the pai- :tpa ratio [ ] . elevated levels of pai- are associated with increased fibrinolysis shutdown, which is the most common fibrinolytic phenotype in the injured population and is associated with an increased mortality due to macro-and microthrombotic complications such as venous thrombotic events (vte) and multiple organ failure (mof) [ ] . fibrinolysis shutdown has previously been identified as the most common fibrinolytic phenotype following injury and is also associated with increased mortality compared to physiologic fibrinolysis, often due to multiple organ failure [ ] [ ] [ ] and has most commonly been measured by thrombelastography (teg), a viscoelastic assay that provides a comprehensive assessment of clot formation and clot remodeling and degradation. therefore, we hypothesize that alcohol intoxication is associated with an increased incidence of fibrinolysis shutdown on thrombelastography. our prospective trauma activation protocol (tap) registry includes all adult (≥ years old) patients who met the criteria for the highest level of trauma team activation from to at the ernest e moore shock trauma center at denver health (dhmc), denver, co, an american college of surgeons verified and state-certified level trauma center. exclusion criteria were unsalvageable injuries (defined by patients in asystole at emergency department arrival), isolated gunshot wounds to the head, pregnancy, documented chronic liver disease, or a known coagulation disorder. a rapid thrombelastogram (r-teg) was run on whole blood from all of these patients under waiver of consent at the scene or immediately upon arrival less than h postinjury. this clinical study was approved by the colorado multiple institutional review board (comirb). clinical data were collected by trained research professional assistants (pras) and included demographic characteristics, injury severity, physiologic derangement, transfusions, and outcomes [death, intensive care-free days (icufd), ventilator-free days (vfd)]. severe traumatic brain injury was defined as an abbreviated injury scale (ais) for head ≥ . the new injury severity score (niss) measured injury severity. massive transfusion was defined as greater than units of red blood cells (rbc) or death within h postinjury as we have found this definition to be related to adverse outcomes [ , ] . further, the inclusion of death within h was to minimize survivor bias (i.e., nonsurvivors did not have the "opportunity" to receive transfusions) [ , ] . the protocol for massive transfusion of blood products has been described previously and includes initial empiric blood component therapy (ffp:rbc in : ratio) [ ] followed by r-teg-guided hemostatic resuscitation base on act [s], angle (°), maximum amplitude [ma (mm)], and lysis min after ma (ly [%] [ ] . samples were collected during trauma activations within h of injury in the field or emergency department (ed) in tubes containing . % citrate. all blood samples were obtained prior to administration of plasma or tranexamic acid (txa). determination of blood alcohol level (bal) was requested at the attending physician's discretion. the lower limit of detection for bal is mg/dl ( . g/l). a team of trained professional research assistants completed the viscoelastic assays. citrated blood samples were analyzed using the teg thrombelastography hemostasis analyzer (haemonetics, niles, il, usa) as previously described [ ] . the rapid teg (activated by tissue factor and kaolin) was employed and the following indices were obtained from the tracings of the teg: activated clotting time [act (s)], angle (°), maximum amplitude [ma (mm)], and lysis min after ma [ly (%)]. definitions for fibrinolysis phenotypes by rteg were shutdown (≤ . %), physiologic (> . -≤ . %), and hyperfibrinolysis (≥ %) as previously described [ ] . sas version . (sas institute, inc. cary, nc, usa) was used for statistical analysis. non-normally distributed variables were expressed as median and interquartile range (iqr) and the wilcoxon non-parametric test or the kruskal-wallis test was used for continuous variables. normally distributed variables were presented as mean (standard deviation, sd). fisher's exact test was used for categorical variables. the non-parametric spearman rho test was used for correlations. receiver operator characteristic (roc) curve analysis was done to determine the predictability of bal to predict fibrinolysis phenotypes. multinomial logistic regression models were used to assess the independent effect of bal on the three lysis phenotypes. model fit was assessed via deviance and the pearson goodness of fit tests (higher p values reflect better fit). to minimize selection bias due to missing bal, we conducted a sensitivity analysis including the patients without a measured bal as an additional, separate category. all tests were two tailed with significance declared at p < . . of consecutive trauma activation patients enrolled between and , ( . %) had blood alcohol levels (bal) measured. table illustrates differences in patients that had a bal measured and those that did not. compared to patients for whom bals was not obtained, those with bals measured were more likely to have blunt trauma ( . % vs . %, p < . ) and tbi ( . % vs. . %, p = . ), had increased incidence of isolated tbi ( . % vs . %, p = . ), had slightly, but significantly lower inr ( . ( . ) vs. . ( . ), p = . ), were less likely receive antifibrinolytics in the form of tranexamic acid ( . % vs . %, p = . ) and were less likely to undergo a massive transfusion ( . % vs . %, p < . ). other demographic and injury characteristics were similar. the median bal was mg/dl ( . g/l) (iqr: - ); therefore, we used this cutoff to define the high bal group (> mg/dl or > . g/l) versus low bal ( - mg/dl or . - . g/l). the third category was undetectable bal (< mg/dl or < . g/l). overall, ( %) patients had no detectable bal, ( . %) had bal of - mg/dl ( . - . g/l), and ( . %) patients had bal > mg/dl (> . g/l). characteristics of patients stratified by bal categories are depicted in table . patients with a high bal had lower admission systolic blood pressure (sbp), increased base deficit (bd), and decreased ly (p < . for all) ( table ) . bal had a moderate, but significant inverse linear correlation with ly (rho = − . , p < . ), while there were no significant correlations between bal and other teg values (act, angle, ma) (table ). the distribution of fibrinolysis phenotypes varied significantly by bal levels (fig. , p = . ), with high bal having more shutdown and less hyperfibrinolysis than the other two bal groups. multinomial logistic regression for the three-category lysisdependent variable (hyperfibrinolysis, shutdown, and physiologic serving as the reference group) showed that, after adjustment for age, blunt mechanism, niss, admission gcs, and sbp, bal levels > mg/dl (≫ . g/l) were independently associated with a threefold increase in the odds of shutdown compared to undetectable bal (or . , % ci . - . , p = . ). high bal was also significantly associated with higher odds of shutdown compared to low bal (or . , % ci . - . ) as shown in table . low bal was not significantly associated with abnormal lysis. hyperfibrinolysis was not associated with either high or low bal. a bal > is an independent predictor of fibrinolysis shutdown (auroc . , % . - . ). in the sensitivity analysis, we added the patients for whom bal was not obtained, adjusted for the same covariates as above. high bal remained independently associated with shutdown compared to undetectable bal (or . , % ci . - . ) and compared to untested patients (or . , % ci . - . ). in binomial multiple logistic regression, high bal did not independently affect mortality (p = . ), vfd < (p = . ), or icufd < days (p = . ), nor did it modify the association between fibrinolysis phenotype and these outcomes (p > . for all interactions). we confirmed previous results that compared to physiologic fibrinolysis, both abnormal fibrinolysis phenotypes were associated with increased mortality (hyperfibrinolysis or . , % ci . - . ; shutdown or . , % ci . - . ) and vfd < (hyperfibrinolysis or . , % ci . - . ; shutdown or . , % ci . - . ). the number of patients with high bal who died (n = ) and the number of vte in these patients (n = ) were too small to allow reliable analyses within these subgroups. the distribution of fibrinolysis phenotypes in intoxicated trauma patients was shifted toward an increased prevalence of fibrinolysis shutdown compared to patients with no detectable alcohol. at the same time, this did not result in an increased risk of death. previous studies have indicated a number of perturbations in viscoelastic-based measurements of blood clotting in an intoxicated and injured patient [ , ] . two previous studies have each shown that the time to clot formation and rate of clot propagation are impaired in intoxicated patients. our study does not show significant increases in the time to clot formation as measured by the activated clotting time (act) in intoxicated patients or the rate of clot propagation. an explanation could be the type of activator used for the teg assays. both previous studies used kaolinactivated tegs while our institution uses rapid tegs, which are activated with tissue factor as well as kaolin. our results are consistent with findings in a recent study [ ] suggesting that major adverse outcomes (need for massive transfusion and death) are not significantly affected by acute alcohol intoxication. howard et al. [ ] showed no difference in rates of transfusion and death in those who were intoxicated, specifically, the rates of death and massive transfusion are similar regardless of bal class for each fibrinolysis phenotype. overall, we found that patients who had bal measured seemed slightly more likely to have tbi but had less bleeding/shock than those that were not tested for bal. this could be explained by the fact that alcohol intoxication suppresses cognitive and motor function resulting in a lower gcs. several studies have investigated the potential role of alcohol consumption and thromboembolic risk. spoerke et al. [ ] showed that both males and females had increased pai- , the inhibitor of tpa-mediated fibrinolysis, after consumption of alcohol. in the male population, this was also associated with a small decrease in ly (clot breakdown). in a study evaluating the effects of alcohol consumption on cardiac risk, djousse et al. [ ] found that individuals who consumed larger amounts of alcohol, more than . g of alcohol per day (the equivalent of one drink), had increased circulating pai- , which they postulated increased the risk of thrombotic complications. furthermore, evidence supports that in acute alcohol intoxication, there is an increase in both tpa and pai- [ , ] . however, the ratio of pai- :tpa is shifted to favor pai- with the consumption of an increased amount of alcohol [ ] . furthermore elevated triglyceride levels resulting from heavy alcohol consumption may further stimulate pai- gene expression, especially in people with a genetic makeup particularly sensitive to pai- [ ] . this increased pai- gene expression could result in the inhibition of fibrinolysis and thus increase the risk for acute cardiac events [ ] . interestingly, the type of alcohol consumed appears to effect the ratio of pai- :tpa. tousoulis et al. investigated the effects of types of alcohol on the fibrinolytic system and discovered that acute alcohol consumption increased the pai- :tpa ratio; however, this effect was not observed after the consumption of red wine [ ] . these data support the notion that not only the amount of alcohol consumed but the type may adversely affect cardiovascular health. in our study, a higher bal (> mg/dl or > . g/l) was associated with a lower ly on r-teg suggesting increased resistance to tpa in this population. furthermore, the odds of fibrinolysis shutdown were threefold higher if the bal was > mg/dl (> . g/l) and this bal was also an independent predictor of fibrinolysis shutdown with an auroc of . , indicating bal > mg/dl (> . g/l) is a fair predictor of fibrinolysis shutdown. in vitro and in vivo studies have provided data that ethanol affects the fibrinolysis profile and studies in healthy human volunteers suggest that this decrease in fibrinolysis is secondary to patients with a high bal class had an increased incidence of fibrinolysis compared to those with no detectable blood alcohol and those with > - mg/ dl ( - . g/l) circulating levels of pai- [ , ] . our data are consistent with other previously published data that high levels of alcohol intoxication are associated with decreased fibrinolytic activity while low level of alcohol intoxication is not [ ] [ ] [ ] [ ] . furthermore, these data confirm that of howard et al. who also recently evaluated alcohol effects on fibrinolysis using rotem [ ] . they similarly illustrated that elevated levels of etoh resulted in decreased fibrinolysis by rotem [ ] . the confirmation of impaired fibrinolysis, on two similar but different viscoelastic platforms, in these two separate patient populations strengthens the notion that acute alcohol intoxication influences fibrinolytic phenotype more than previously thought and could be a risk factor for the development of fibrinolysis shutdown-related adverse outcomes. several studies have evaluated distinct fibrinolysis phenotypes, with fibrinolysis shutdown being associated with increased mortality and rates of organ failure [ , , ] . the crash- trial suggested that the early empiric use of tranexamic acid (txa) reduces the rate of death in injured patients but identified increased mortality when this therapy was delivered > h after injury [ ] . our group has argued that there should be a selective use of txa in the injured patient as administration to those with a shutdown phenotype may be critical in the pathogenesis of postinjury organ failure and thrombotic complications [ ] . at our institution, txa is administered to patients that are in hemorrhagic shock with evidence of hyperfibrinolysis on teg as we have found that the use of txa in those not in hyperfibrinolysis may increase the risk of postinjury organ failure, thrombotic complications, and mortality [ , ] . experimental data have revealed that ethanol-exposed animals show enhanced pai- expression and pulmonary fibrin deposition with coincident exaggeration of pulmonary edema and inflammatory injury [ ] . high levels of alcohol and subsequent excess expression of pai- may thus be a contributing factor toward the development of organ failure following injury and would likely be exacerbated with the concomitant use of antifibrinolytic therapy. less than % of trauma activation patients received antifibrinolytic therapy overall, and less than % in patients that had a bal measured making subgroup analysis of patients that received txa unreliable. however, while our data reveal increased odds of fibrinolysis shutdown in acutely intoxicated patients, there is an absence of increased mortality in this group as we would expect for those patients in fibrinolysis shutdown. this is also seen in other studies evaluating alcohol's effects on fibrinolysis [ ] . this could be due to an underpowered study to identify mortality differences. furthermore, once the acute episode of intoxication resolves, the fibrinolytic phenotype could change from that of fibrinolysis shutdown to physiologic fibrinolysis or even hyperfibrinolysis. it may be that the patients who remain in a phenotype of fibrinolysis shutdown after the resolution of acute alcohol intoxication are the ones that have adverse effects in the form of late mortality or mof. identifying patients with a high bal would allow the stratification of patients into those with a higher risk of having fibrinolysis shutdown. low fibrinolytic activity has been associated with an increased mortality following injury, with a mortality of - % compared to a physiologic level of fibrinolysis of - % [ , ] . the cause of death in the shutdown cohort was typically late death as previously discussed, and more commonly associated with multiorgan failure [ , ] . it has been a hypothesis that multiple organ failure is caused by microthrombotic complications stemming from the decreased clot breakdown associated with fibrinolysis shutdown [ , ] . there are no pharmacologic interventions currently in use to specifically target fibrinolysis shutdown. however, by identifying alcohol intoxication as a modulator of the fibrinolysis phenotype, this could help guide future interventions to help reduce the incidence of fibrinolysis shutdown and subsequent micro-and macrothormbotic complications in the critically injured patient. data suggest that an increased amount of pai- and shifting the tpa:pai- ratio in favor of pai- is a likely factor that contributes toward increased incidence of fibrinolysis shutdown in the intoxicated patient. the presented study has some limitations. these data reflect a single point in time of a dynamic process and does not take into account the temporal changes of the coagulation process and subsequent evolution of fibrinolysis phenotypes. unfortunately, there were patients in our tap protocol that could not be included in the study because no bal was obtained. bal was obtained at the discretion of the trauma team. this may have led to a selection bias that must be taken into consideration with the interpretation of these results. furthermore, we did not have information on the amount or type of alcohol ingested, the time during which alcohol was consumed, and reliable data on chronic ingestion of alcohol, which may have diverse effects. there were very few patients within our analyzed cohort that had documentation of chronic alcoholism (n = , . %). there was a statistically significant trend (m-h chi-sq, p = . ) of high a bal with documentation of chronic alcoholism, which may be due to increased frequency of documentation among bal-positive patients. the significant association between bal levels and ly was detected again among patients without documentation of chronic alcoholism (p = . ). however, the small number of patients with documented alcoholism limits the analysis in this group and, therefore, for the purpose of this study, we did not separate acute vs chronic users for analysis. we also acknowledge that a high bal leading to increased risk of vte is based on theory at this time as our cohort was not large enough to appropriately study this question. in conclusion, in the injured patient, high bal (> mg/dl or > . g/l) is an independent predictor for fibrinolysis shutdown. the mechanism of alcohol-related shutdown remains to be elucidated however, data would suggest a shift in the pai- :tpa ratio favoring pai- may be a major contributor. postinjury fibrinolysis shutdown has been associated with increased risk of organ failure and thrombotic complications; thus, a high bal may represent a risk factor for vte and microthrombotic events. furthermore, the use of anti-fibrinolytic agents in patients with high bal may have adverse effects by precipitating a more profound resistance to fibrinolysis. author contributions grs implemented the study, interpreted data, drafted and critically revised the manuscript. hbm interpreted data, drafted, and critically revised the manuscript. grn interpreted data, drafted, and critically revised the manuscript. bjh interpreted data, drafted, and critically revised the manuscript. eem, ccs, ab, and as are principal investigators and were responsible for study conception and design, implementation of study, completion of study, interpretation of data, manuscript drafting, and critical revision. conflict of interest gregory r stettler md declares that he has no conflict of interest. ernest e moore md declares that he has no conflict of interest. geoffrey r nunns md declares that he has no conflict of interest. hunter b moore md declares that he has no conflict of interest. benjamin r huebner md declares that he has no conflict of interest. christopher c silliman md, phd, serves on the scientific advisory board for hemanext™. anirban banerjee phd declares that he has no conflict of interest. angela sauaia md, phd, declares that he has no conflict of interest. ethical standards research reported in this publication was supported in part by the national institute of general medical sciences grants: t -gm and p -gm , the national heart lung and blood institute um -hl , in addition to the department of defense usamraa and w xwh- - - . the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health, the national heart, lung, and blood institute, or the department of defense. additional research support was provided by haemonetics with shared intellectual property. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (comirb # - ) and with the helsinki declaration and its later amendments or comparable ethical standards. informed consent was obtained from all individual participants or their surrogates included in the study. alcohol and trauma: the perfect storm effects of ethanol intoxication and gender on blood coagulation the effects of alcohol on coagulation in trauma 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fibrinolysis system ethanol impairs coagulation and fibrinolysis in whole blood: a study performed with rotational thromboelastometry exposing the bidirectional effects of alcohol on coagulation in trauma: impaired clot formation and decreased fibrinolysis in rotational thromboelastometry hyperfibrinolysis, physiologic fibrinolysis, and fibrinolysis shutdown: the spectrum of postinjury fibrinolysis and relevance to antifibrinolytic therapy the crash- trial: a randomised controlled trial and economic evaluation of the effects of tranexamic acid on death, vascular occlusive events and transfusion requirement in bleeding trauma patients rationale for the selective administration of tranexamic acid to inhibit fibrinolysis in the severely injured patient plasminogen activator inhibitor- is critical in alcohol-enhanced acute lung injury in mice key: cord- - rpiv d authors: giantsou, elpis; liratzopoulos, nikolaos; efraimidou, eleni; panopoulou, maria; alepopoulou, eleonora; kartali-ktenidou, sofia; manolas, konstantinos title: de-escalation therapy rates are significantly higher by bronchoalveolar lavage than by tracheal aspirate date: - - journal: intensive care med doi: . /s - - -x sha: doc_id: cord_uid: rpiv d objective: to assess outcomes with de-escalation therapy in ventilator-associated pneumonia (vap). design: prospective observational study. setting: multidisciplinary intensive care unit. patients and participants: vap was diagnosed by positive quantitative cultures of both tracheal aspirate and bronchoalveolar lavage (bal) and treated appropriately for all significant isolates of tracheal aspirate and bal in patients who were assigned to de-escalation therapy by bal or tracheal aspirate. interventions: none. measurements and results: antibiotic therapy was de-escalated in patients ( . %), who had decreased mortality at day ( . % vs. . %) and day ( % vs. . %) and shorter intensive care unit ( . ± . vs. . ± . days) and hospital ( . ± . vs. . ± . days) stay (p < . ). of the patients assigned to tracheal aspirate, the ( %) who achieved de-escalation of therapy had reduced -day mortality ( . % vs. . %), reduced -day mortality ( . % vs. . %), and shorter intensive care unit ( . ± . vs. . ± . days) and hospital ( . ± . vs. . ± . days) stay (p < . ). of the patients assigned to bal, the ( . %) who achieved de-escalation of therapy had decreased -day mortality ( . % vs. . %), decreased -day mortality ( . % vs. %), and shorter intensive care unit ( . ± . vs. . ± days) and hospital ( . ± . vs. . ± . days) stay (p < . ). conclusions: for patients with vap who have had appropriate treatment and shown a favorable clinical response, mortality and duration of stay can be further improved by de-escalation therapy. antibiotic therapy of patients with ventilator-associated pneumonia (vap) is now regarded as a two-stage process [ ] [ ] [ ] . the first stage involves administering broadspectrum antibiotics to avoid inappropriate treatment in patients with true bacterial pneumonia. the second stage involves de-escalating an initially appropriate antibiotic regimen to avoid overuse of antibiotics, by streamlining, shortening or stopping therapy, as dictated by the patient's clinical response and information about the bacteriology of the infection. although de-escalation therapy is suggested to be part of any appropriate antimicrobial stewardship in patients with vap [ ] , controversy continues regarding the outcomes associated with its implementation. indeed, some reports suggest that de-escalation therapy does not affect mortality and duration of intensive care unit (icu) and hospital stay [ , ] , whereas others claim that it does significantly improve them [ , ] . moreover, there are suggestions that de-escalation therapy improves mortality at weeks after vap onset, but does not significantly affect either mortality at month or the duration of icu and hospital stay [ ] . however, the potential effect of de-escalation therapy on outcomes, that are based on clinical diagnosis of vap, may be difficult to assess, as clinical diagnosis is frequently associated with risk for overestimation of the incidence of infection [ , ] . therefore, the objective of this study was to assess outcomes in association with the implementation of de-escalation therapy in a well-defined group of patients [ ] who had developed vap as confirmed by two separate cultures, one quantitative tracheal aspirate and one bronchoalveolar lavage (bal) sample, that both had to be positive. the study was conducted during a -month period at the university of thrace teaching hospital ( beds), greece. patients were entered into the study if: (a) they were older than years of age and (b) their physicians established a diagnosis of vap. patients were excluded if they (a) were temporarily transferred to our icu due to lack of available beds in another hospital, (b) had received solid organ or bone marrow transplant or (c) had human immunodeficiency virus infection. the study was approved by the local human studies ethics committee, and informed consent was obtained as appropriate. the initial antibiotic regimen was de-escalated when the patient's clinical response and the microbiological information permitted. the clinical response was decided at h reassessment by absolute consensus of five attending physicians after a clinical round. the microbiological information that the attending physicians used to de-escalate antibiotic therapy, to establish the appropriateness of the initial antibiotic regimen and to confirm the diagnosis of vap, in patients with clinical suspicion for infection, was derived from quantitative tracheal aspirate or bal. nevertheless, because the aim of the study was to assess outcomes with de-escalation therapy and not to evaluate the contribution of different methods to diagnosis, we studied only patients in whom the diagnosis of vap was confirmed by both quantitative tracheal aspirate and bal samples, and who received an appropriate empiric antibiotic regimen for all isolates of tracheal aspirate and bal that reached significant concentration. data interpretation was performed by two independent investigators and the attending physicians were blind to the nature of the study. patients could not be entered more than once into the study, and only the first vap episode for each patient was considered. the type of respiratory sample that guided deescalation therapy, quantitative tracheal aspirate or bal, was determined by the availability of services. a respiratory sample collection kit (tracheal aspirate or bal) was delivered to icu every h and was assigned to the first patient in need. using the kit assigned to the case, the attending physicians collected the respiratory sample within h after clinical suspicion for vap was raised. patients in whom a kit other than the assigned kit was used were not studied. however, all patients had both types of samples, because, within h after the collection of tracheal aspirate or a bal sample by the attending physicians, the other type of sample was collected by two independent investigators before any antibiotic introduction or modification. the empiric antibiotic regimen was started as soon as both samples, tracheal aspirate and bal, had been collected. tracheal aspirates and bal were both cultured with quantitative technique. the clinical suspicion for vap was raised according to predefined criteria [ ] . late-onset vap was developed after at least days of mechanical ventilation [ ] . de-escalation therapy was defined as either the switch to an agent that was less broad spectrum than initial therapy, or the use of fewer drugs [ ] . thus it was possible to have either or both types of antibiotic changes in a given patient. the use of fewer drugs involved stopping oxazolidinone when mrsa was not grown, and aminoglycoside or quinolone when p. aeruginosa was not identified. the empiric antibiotic regimen was guided by the gram stain, the presence of risk factors for multiresistant pathogens and the local microbiology data [ ] . antibiotics were ranked according to activity spectrum against gram-negative bacteria (highest , lowest ) as follows [ , ] : carbapenems ; extended spectrum penicillins , quinolone and aminoglycoside , and beta-lactams [ ] . this rank of activity spectrum was used to de-escalate therapy to the narrowest spectrum agent available. in the absence of p. aeruginosa, carbapenem was de-escalated to extendedspectrum penicillin, or to quinolone, or if possible to a non-antipseudomonal beta-lactam, and extendedspectrum penicillin was de-escalated to quinolone or if possible to a non-antipseudomonal beta-lactam. in the presence of p. aeruginosa, de-escalation therapy was as above but, the final regimen had to include two antipseudomonal drugs. therefore, in this case carbapenem was de-escalated to extended-spectrum penicillin or to a antipseudomonal beta-lactam, and extended-spectrum penicillin to an antipseudomonal beta-lactam, while quinolone or aminoglycoside was continued as part of the combination regimen. during the study period clinical suspicion for vap was raised in patients, of whom ( . %) were diag-nosed with vap by two positive cultures, one quantitative tracheal aspirate and one bal. of these, ( . %) had received appropriate initial treatment, confirmed by both tracheal aspirate and bal, and demonstrated favorable clinical response. the first available respiratory sample collection kit, tracheal aspirate or bal, was used to collect the sample that guided de-escalation therapy in ( . %) patients, of whom ( . %) had de-escalation therapy according to the predefined protocol and finally constituted the study population. on admission the following variables were recorded: age, gender, simplified acute physiologic score (saps) ii [ ] , sequential organ failure assessment (sofa) score [ ] , admission category (medical versus surgical, elective versus emergency surgical), origin (medical or surgical ward versus home) indication for mechanical ventilation and predicted mortality according to saps ii. at collection of respiratory samples, the following baseline variables were recorded: previous duration of mechanical ventilation, use of antimicrobials days before vap, temperature in°c, white blood cell count per mm , ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao /fio ), [ ] . before de-escalation of antibiotics we recorded the initial empiric regimen and the pathogens recovered at significant concentrations at the respiratory specimen that guided de-escalation therapy. the primary end point was to assess outcomes with deescalation therapy, in patients with vap, as confirmed by two separate positive cultures, one quantitative tracheal aspirate and one bal. secondary end points were to evaluate outcomes with de-escalation therapy guided by bal and by quantitative tracheal aspirate. descriptive analysis was performed. the number of organisms recovered from tracheal aspirate and bal cultures was expressed as colony-forming units/ml. all other results were expressed as mean ± standard deviation (sd) or as percentages of total values. frequencies were compared by means of a chi-square test with yates correction or fisher's exact test when appropriate. student's t-test was used to compare the means of bal and tracheal aspirate groups. all p-values were two sided and considered significant when they were less than . . a total of patients with vap were prospectively evaluated. the mean age was . ( ± ) years; ( . %) were men. the mean saps ii and sofa score on admission were . ( ± . ) and . ( ± . ) respectively. vap was late-onset in ( . %) of cases. the case mix was medical in %, emergency surgery ; percentages represent patients who initially received the antibiotics presented and in whom were isolated at significant concentrations the pathogens presented, in the quantitative tracheal aspirate or bronchoalveolar lavage sample, that guided de-escalation therapy decisions. percentages may not add up to because of combination multi-drug regimens and isolates with more than one pathogen grown at significant concentration table antibiotics initially prescribed and pathogens identified in the respiratory sample that guided de-escalation therapy decisions in patients with ventilator-associated pneumonia in . % and elective surgery in . %. de-escalation of antibiotic therapy was accomplished in ( . %) patients. no significant differences were noted between patients in whom therapy was de-escalated and those in whom it was not, in admission (table ) and baseline (table ) characteristics, in antibiotics initially prescribed (table ) and in pathogens identified at significant concentrations in the sample (quantitative tracheal aspirate or bal) that guided de-escalation therapy ( table ) . patients in whom antibiotic therapy was de-escalated, compared with those in whom it was not, had reduced day and -day mortality (table ) . of the patients who were assigned to bal, ( . %) achieved de-escalation of antibiotic therapy and, compared with those who did not, despite their similar assignment, had significantly reduced -day and -day mortality (table ) . similarly, of the patients who were assigned to quantitative tracheal aspirate, ( %) achieved de-escalation of antibiotic therapy and, compared with those who did not, despite their similar assignment, had reduced -day and -day mortality ( table ) . patients who achieved de-escalation of therapy had shorter duration of stay in icu and in hospital than those who did not (table ). patients assigned to bal mssa, methicillin-sensitive staphyloccoccus aureus; mrsa, methicillin-resistant staphylococcus aureus; l, linezolide; m, meropenem; a, amikacin; q, quinolone; pt, piperacillin/tazobactam; ctz, ceftazidime; *these mrsa were not taken into account in the pathogens associated with de-escalation of therapy, for only the antibiotic against p. aeruginosa was de-escalated and therefore the only pathogen associated with de-escalation of therapy was p. aeruginosa or quantitative tracheal aspirate in whom treatment was de-escalated, compared with those in whom it was not, despite their similar assignment, had significantly shorter duration of stay in icu and hospital ( the whole group of patients assigned to bal, those who achieved de-escalation of therapy and those who did not, compared with the whole group of those assigned to quantitative tracheal aspirate, had reduced -day mortality ( . % vs. . %, p = . ), reduced -day mortality ( . % vs. . %, p = . ) and fewer days in icu ( . ± . vs. . ± . , p = . ) and in hospital ( . ± . vs. . ± . , p = . ). no significant differences in mortality at day and day or in duration table pathogens and antibiotic agents not associated with de-escalation of antibiotic therapy, in a prospective cohort of patients with ventilator-associated pneumonia pathogens not associated antibiotics not de-escalated patients in whom with de-escalation therapy initial final treatment was not de-escalated, n mssa, methicillin-sensitive staphyloccoccus aureus; mrsa, methicillin-resistant staphylococcus aureus; l, linezolide; m, meropenem; a, amikacin; q, quinolone; pt, piperacillin/tazobactam; ctz, ceftazidime of stay in icu and in hospital were noted between patients in whom de-escalation of therapy was achieved by bal and those in whom it was achieved by quantitative tracheal aspirate (table ) . de-escalation of antibiotic therapy by quantitative tracheal aspirate (table ) comprised: reduction in spectrum in . % ( of patients), for p. aeruginosa; fewer drugs coupled with reduction in spectrum in . % ( of patients). the combined drug number and spectrum reduction was due to cessation of the antistaphylococcal agent, coupled with spectrum reduction for p. aeruginosa, in five patients and with cessation of combination therapy and spectrum reduction, for serratia marcescens that was identified instead of p. aeruginosa, in the rest. de-escalation of antibiotic therapy by bal (table ) comprised: reduction in spectrum in . % ( of patients) for p. aeruginosa; fewer drugs in . % ( of patients) as combination therapy was replaced by monotherapy for klebsiella that was identified instead of p. aeruginosa; fewer drugs coupled with reduction in spectrum in . % ( of patients). the combined drug number and spectrum reduction was due to cessation of the antistaphylococcal agent coupled with spectrum reduction for p. aeruginosa in patients. in the rest, it was due to cessation of combination therapy for the anticipated p. aeruginosa that was not recovered coupled with spectrum reduction for the pathogens identified. de-escalation of therapy by tracheal aspirate was not accomplished (table ) when there were not narrower spectrum agents available for the recovered, as anticipated, pathogens: mrsa and p. aeruginosa in . % ( of ), mrsa alone in . % ( of ), streptococcus or enterococcus in . % ( of ) that was sensitive to oxazolidinone only, p. aeruginosa with proteus in . % ( of ) and other than p. aeruginosa gram-negative bacteria in . % ( of ). in these latter cases two species were cultured, of which one was sensitive only to meropenem and the other only to quinolone or aminoglycoside, preventing de-escalation of the initial combination regimen. de-escalation of therapy by bal was not accomplished (table ) when there were no narrower-spectrum agents available for the recovered, as anticipated, p. aeruginosa. in this study, we observed that -day and -day mortality could be further decreased by de-escalation of antibiotic therapy for patients with vap who were appropriately treated and had favorable clinical response. improvement in icu and hospital duration of stay was also established for patients who get de-escalation therapy. to the best of our knowledge, only a few studies have evaluated the effect of de-escalation therapy on outcomes in patients with vap [ ] [ ] [ ] [ ] [ ] and all have used clinical diagnosis, supported, when available, by cultures of quantitative tracheal aspirate or bal, with the inherent risk for overestimation of the incidence of infection [ , , ] . recently, kollef et al. found reduced mortality for patients who received de-escalation therapy compared to those who did not ( % vs. . %) [ ] . improved mortality was also observed by rello et al. with de-escalation of antibiotic therapy ( . % vs. . %) [ ] and by soo hoo et al ( % vs. %) [ ] . the diagnosis of vap by two positive quantitative cultures, one tracheal aspirate and one bal sample, in all study patients in our data, should probably be considered for the interpretation of the difference between our observations and those of ibrahim et al. and of micek et al., who found no benefits in mortality and duration of stay from de-escalation therapy [ , ] . at least two factors may explain why patients in whom treatment was de-escalated had significantly reduced day and -day mortality, compared with patients in whom it was not, despite the absence of significant differences in admission and baseline characteristics, predicted mortality, prescribed antibiotics and identified pathogens for the two groups. first, patients in whom antibiotic therapy was deescalated ended up receiving fewer antibiotics. stopping unnecessary antibiotics helps to prevent potentially harmful side effects and has been shown to reduce airway colonization [ ] [ ] [ ] and the risk of secondary episodes of infection [ , ] , which are known to adversely affect patient outcome [ , ] . within this context should probably be interpreted the good outcomes that we observed, in consistency with previous reports [ ] , for patients assigned to management by bal, as more than half of them had de-escalation of antibiotic therapy and therefore ended up receiving fewer unnecessary antibiotics. second, patients in whom antibiotic therapy was deescalated had shorter exposure to acute-care settings, as they had shorter duration of icu and hospital stay. contact with such settings [ ] has been suggested to be among the factors that are likely to influence the potential for resistance of pathogens identified in vap [ , , ] . re-ducing duration of exposure to an acute-care setting may help to decrease exposure to several procedures and therapies that are known to modify host defenses [ , ] , in the midst of a steady selection pressure for multi-resistant microorganisms [ ] , which are well known to adversely affect patient outcome [ , ] . our study was limited by its performance within a single icu and the relatively large subset of patients that was excluded, namely those in whom de-escalation therapy was not according to the protocol and those in whom the first available service for respiratory sample collection was not used. in addition, the diagnosis of vap and the appropriateness of the empiric treatment had to be confirmed by both quantitative tracheal aspirate and bal. thus, the results of this study cannot necessarily be extended to other populations. another limitation is that investigators were aware of the patients' assignment and of their de-escalation therapy status. however, obstacles in blinding were posed by the necessity to collect both samples in all study patients and to modify treatment in the context of de-escalation therapy. nevertheless, our efforts were focused on standardizing care and using rigorous criteria to evaluate outcomes. finally, it is important to acknowledge that our study was not specifically designed to test the hypothesis that de-escalation therapy is superior to a non-de-escalation regimen in terms of improving clinical outcomes. only a prospective randomized trial comparing these two approaches would be able to answer such a question. in summary, for patients with vap who were appropriately treated and had favorable clinical response, we found that mortality and duration of icu and hospital stay could be further reduced by de-escalation therapy. this finding provides arguments for stopping overuse of antibiotics when alternative regimens, with narrower-spectrum or fewer antibiotics, are available. conference summary: ventilator associated pneumonia the importance of de-escalating antimicrobial therapy in patients with ventilatorassociated pneumonia antibiotic prescribing for ventilator-associated pneumonia: get it right from the beginning but be able to rapidly deescalate experience with a clinical guideline for the treatment of ventilator-associated penumonia a randomized controlled trial of an antibiotic discontinuation policy for clinically suspected ventilator-associated pneumonia clinical characteristics and treatment patterns among patients with ventilator-associated pneumonia de-escalation therapy in ventilator-associated pneumonia impact of clinical guidelines in the management of severe hospital acquired pneumonia value of the clinical pulmonary infection score for the identification and management of ventilator-associated pneumonia clinical pulmonary infection score for ventilator-associated pneumonia: accuracy and inter-observer variability comparison of vs. days of antibiotic therapy for ventilatorassociated pneumonia in adults guidelines for the management of adults with hospital-acquired, ventilator-associated and healthcareassociated pneumonia gibert c ( ) ventilator-associated pneumonia caused by potentially drugresistant bacteria de-escalation therapy in ventilator-associated pneumonia de-escalation in lower respiratory tract infections escalation/deescalation of initial empiric ventilatorassociated pneumonia therapy: interim results from the assessment of local antibiotic resistance measures study a new simplified acute physiology score (saps ii) based on a european/north american multicenter study the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine year in review in intensive care medicine, . ii. infection and sepsis, ventilator-associated pneumonia, ethics, haematology and haemostasis, icu organization and scoring, brain injury diagnosis and treatment of ventilator-associated pneumonia: fiberoptic bronchoscopy with bronchoalveolar lavage is essential invasive and noninvasive strategies for management of suspected ventilator-associated pneumonia resolution of infectious parameters after antimicrobial therapy in patients with ventilator-associated pneumonia short course empiric antibiotic therapy for pulmonary infiltrates in the intensive care unit: a proposed solution for indiscriminate antibiotic prescription therapy of ventilator-associated pneumonia: what more can we do to use less antibiotics? both early-onset and late-onset ventilator-associated pneumonia are mainly caused by potentially multiresistant bacteria a comparative analysis patients with early vs. late onset nosocomial pneumonia in icu setting patterns of colonization by pseudomonas aeruginosa in intubated patients: a -year prospective study of , isolates using pulsed-field gel electrophoresis with implications for prevention of ventilator-associated pneumonia preventing ventilator-associated pneumonia: an evidence-based approach of modifiable risk factors biofilms, antimicrobial resistance and airway infection ventilatorassociated pneumonia the importance of a de-escalating strategy for antibiotic treatment of pneumonia in the icu key: cord- -h vcmrd authors: mikulska, malgorzata; furfaro, elisa; de carolis, elena; drago, enrico; pulzato, ilaria; borghesi, maria lucia; zappulo, emanuela; raiola, anna maria; grazia, carmen di; del bono, valerio; cittadini, giuseppe; angelucci, emanuele; sanguinetti, maurizio; viscoli, claudio title: use of aspergillus fumigatus real-time pcr in bronchoalveolar lavage samples (bal) for diagnosis of invasive aspergillosis, including azole-resistant cases, in high risk haematology patients: the need for a combined use with galactomannan date: - - journal: med mycol doi: . /mmy/myz sha: doc_id: cord_uid: h vcmrd diagnosis of invasive aspergillosis (ia) is challenging, particularly in high-risk patients with lung lesions other than typical according to -eortc/msg criteria. even if microbiology is positive, they still remain unclassified according to -eortc/msg. quantitative polymerase chain reaction (qpcr) provides new mycological documentation of ia. this retrospective study assessed aspergillus fumigatus real time qpcr (mycogenie®) in bal to diagnose ia and identify azole-resistant strains. clinical, radiological, and microbiological data from hematology patients ( % hsct recipients; % on mould active agents) from years - were collected; and bal samples were tested with qpcr (cutoff: ct < ) and galactomannan (gm, platelia®, cutoff: . odi). patients were classified as proven/probable, possible, and no-ia. "atypical-ia" referred to patients with lesions other than typical according to -eortc/msg and positive mycology. proven ia was diagnosed in two cases ( . %), probable in ( . %), possible in ( %), atypical in ( . %). qpcr was positive in samples ( . %). sensitivity and specificity of qpcr for proven/probable ia (vs no-ia; atypical-ia excluded) were % ( % confidence interval [ci]: – ) and % ( %ci: – ), respectively. sensitivity of qpcr was higher when combined with gm ( %, %ci: – ) and in those receiving mould-active agents at bal ( %, %ci: – ). one sample had tr /l h mutation. in conclusion, in high-risk hematology patients with various lung lesions, a. fumigatus qpcr in bal contributes to diagnosing ia, particularly if combined with gm and in patients receiving mould-active agents might allow detecting azole-resistant mutations in culture negative samples. invasive fungal disease (ifd), and particularly invasive aspergillosis (ia), is an infectious complication affecting mainly patients with haematological disorders and prolonged neutropenia, long-term high dose corticosteroid treatment or allogeneic stem cell transplantation (sct). [ ] [ ] [ ] ia in this setting is associated with high morbidity and mortality, particularly when not promptly diagnosed and treated. unfortunately, the diagnosis of ia in hematology patients is challenging because of nonspecific clinical manifestations, low yield of fungal cultures, and difficulty in performing invasive diagnostic procedures due to thrombocytopenia or poor general conditions. in addition, azole resistant a. fumigatus strains have been increasingly frequent in several geographical regions, and given low rate of positive cultures, these cases risk to remain undetected, compromising the outcome of ia. diagnostic criteria for ifd in the immunocompromised patients have been developed by eortc/msg in and subsequently revised in . they established three levels of certainty of diagnosis: proven, probable, and possible. in particular, for probable ia, a combination of a host factor (presence of a predisposing condition) plus a clinical criterion plus a mycological criterion are required. clinical criteria in cases of pulmonary ia include one of the following typical radiological lesions in lung computed tomography (ct) scan: ( ) dense, well-circumscribed lesions(s) with or without a halo sign, ( ) air-crescent sign, or ( ) cavity. mycological criteria for pulmonary aspergillosis are detection of galactomannan (gm) in serum or bronchoalveolar lavage fluid (bal) or direct tests positive in sputum or bal. possible ia is diagnosed in the presence of host and clinical criteria but in the absence of mycological documentation. although these criteria revolutionized the clinical research and epidemiological studies in ia, they do not cover numerous possible clinical situations. among them, the most frequent is the presence of host criteria with positive mycological criteria and lung lesions, which are different from the aforementioned typical ones. these patients remain "unclassified" according to eortc/msg criteria, but they are usually treated for ia, and several studies showed that they truly have ia. in fact, in a recent observational eortc study, they were classified as those in whom ifd cannot be excluded, thus not suitable for being considered certain negative controls (european prospective invasive mould disease audit [pimda] protocol). additionally, the performance of gm might be suboptimal in certain settings, for example, in patients receiving mould active agents, in whom breakthrough ia is suspected. molecular methods such as polymerase chain reaction (pcr) are able to detect aspergillus dna in bal samples with good sensitivity ( . %) and specificity ( . %). although in recent years many publications [ ] [ ] [ ] focused on the diagnostic role of aspergillus pcr, its use is not yet recommended in the update of idsa guidelines on the diagnosis and management of aspergillosis, mostly because of the lack of standardised and validated assays. , their advantages include the possibility to detect fungal dna also if it is no longer viable, such as after antifungal treatment has been started, and to confirm the presence of aspergillus with higher sensitivity than culture, similarly to gm. additionally, certain molecular methods offer the possibility to detect resistance mutations in aspergillus fumigatus even in the absence of strain's growth. the aim of the study is to evaluate the performance of a commercially available aspergillus fumigatus real-time quantitative pcr (qpcr), alone and in combination with gm, in bal samples form patients at high risk of ia, and with various radiological lesions, including those receiving mould active antifungals. additionally, the rate of mutations conferring azole resistance in high risk patients in our center was also evaluated. the study was conducted in ospedale policlinico san martino, a tertiary care center in genoa, italy, with active allogeneic sct center. we retrospectively identified all available bal samples from years - from patients with sct and/or haematological malignancies. all patients had pulmonary lesions reported on ct scan. bal samples from the same patient were excluded if performed within weeks. all patients underwent at least two determinations of gm (platelia tm bio-rad-inc.) and , -beta-d-glucan (bdg) (fungitell r assay) in serum, with cutoff values for positivity of . optical density index (odi) and pg/ml, respectively, according to manufactures' recommendations. all bal samples were subject to the following analyses: culture for bacteria, filamentous fungi and mycobacteria, gm, molecular testing for pneumocystosis, mycobacterium tuberculosis, cytomegalovirus, herpes simplex virus, and respiratory viruses (influenza, parainfluenza, respiratory syncytial virus, metapneumovirus, enterovirus, rhinovirus, coronavirus) and bacteria (legionella, bordetella, streptococcus pneumoniae, haemophilus influenzae, chlamydia, mycoplasma pneumoniae). direct research for hyphae and antifungal susceptibility testing were not performed routinely. for bal gm testing, samples were centrifuged and μl of supernatant was further treated according to manufacturer's instruction. residual sample after gm testing was stored at − • c. for gm the cutoff value of . odi was considered positive according to manufacturer's instruction. for each patient, general data, clinical characteristics, data on the underlying disease, including sct, administration of mould active antifungals, and outcome were collected. all patients gave informed consent for data collection and research purposes at the hospital admission and bal performance. the study was approved by local ethics committees under the reference number pr reg . for all patients, microbiological results and full medical records were reviewed. ct lesions were revalued by two radiologists (i.p. and g.c.) with an expertise in pulmonary fungal infections and classified as typical ifd lesions according to eortc/msg revised criteria, or other (atypical) lesions. considering also mycological criteria, patients were stratified according to eortc/msg criteria into four groups: ( ) proven/probable ia, ( ) possible ia, ( ) atypical lung lesions but positive mycological criteria, considered as atypical ia, ( ) no ia with atypical lung lesions and negative mycological criteria. these groups were mutually exclusive but grouped together for the purposes of analyses. the evaluations of qpcr performance were carried out considering as cases patients with proven/probable/possible ia (group and ) or proven/probable ia (group ) or proven/probable ia/atypical ia (atypical lesions with mycology positive, group and ), and considering as controls patients from group . the performance of qpcr was evaluated separately for those receiving and not receiving mould active antifungals (either as prophylaxis or treatment) at the time of bal. bal samples were stored at − • c and subsequently sent to institute of microbiology at università cattolica del sacro cuore in rome where molecular diagnostic methods have been performed. they were tested with aspergillus fumigatus real-time qpcr assay mycogenie r (ademtech, pessac, france). this is a multiplex commercially available ce-ivd marked assay approved for testing respiratory samples which detects dna by targeting the s rrna multicopy gene and specific tr /l h mutations in the cyp a single copy gene of a. fumigatus. dna was extracted with a mycogenie r dna extraction kit automag solution indicated for the isolation and purification of fungal dna. an amount of microliters of stored bal fluid was centrifugated at , rpm for min, after removing microliters of the supernatant, the pellet was resuspended with microliters of tissue lysis buffer and microliters were used to perform the dna extraction in an automag solution instrument equipped with a magnetic particle processor for dna purification kits (ademtech). samples were eluted in microliters. an internal extraction control was added together with the samples during the extraction process as indicated in the manufacturer procedure assay. positive and negative pcr controls were added for each pcr experiment. the kit performance data fixed the limit of detection (lod) of monocopy sequences (tr and l h cyp a mutations) was determined at six copies. for multicopy genes (aspergillus rrna gene), the lod is below one copy. the specificity of primers and probes was verified by the manufacturer and indicated as highly specific ( %). the cutoff for positivity was considered as < (cycle threshold) ct. the performance of cutoff ≤ ct was also evaluated. the variables were reported as median value with range. sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) were calculated where applicable and reported with % confidence intervals ( % ci). distribution of continuous and categorical variables were evaluated with, respectively, mann-whitney and χ test or fisher exact test if applicable. statistical analysis was performed using medcalc's diagnostic test evaluation calculator ( c medcalc software bvba). a total of bal samples from patients were collected. in patients, data from a second bal procedures, performed in median days after the first one (range, - days), were also included in the study. the patients' characteristics are reported in table . briefly, ( . %) of them were male, median age was years (range, - years) and the most frequent underlying disease was acute myeloid leukaemia ( . %). in ( . %) cases, bal was performed after sct, allogeneic in % of cases, in median days after sct (range, - days); and in cases as diagnostic work up in pre-sct evaluation, in median days before sct. at the time of bal, patients ( . %) were neutropenic and ( . %) were receiving mould active agents. ct scan was performed in median days before bal. according to eortc/msg criteria, proven ia was diagnosed in two cases ( . %), probable ia in ( . %), and possible in ( %). overall, / probable cases had positive gm in bal, while in seven cases, all receiving antifungal therapy, the diagnosis of ia was made with serum gm in median days before bal, which was performed mainly due to suspected failure or breakthrough infection. among the remaining patients without eortc/msg typical lesions, had a positive mycological result: gm in bal in cases, with median odi of . (range, . - . ) and serum gm in one case, and were considered cases of atypical ia. in seven cases ( . %) bal cultures were positive for filamentous fungi: five aspergillus (three a. flavus, one a. fumigatus, one a. niger), one fusarium spp., and one paecilomyces spp. patients with growing aspergillus were diagnosed with proven/probable ia in four cases, and atypical ia in one. in bal samples gm was positive, with median odi of . (range, . - . ). aspergillus fumigatus qpcr was positive in samples with median cycle to positive of . (range, - ). the concordance between typical radiological lesions, gm positivity in bal and qpcr positivity was limited, as shown in figure , both in patients receiving mould active agents and in patients without ongoing therapy. also irrespective of radiological lesions, there was poor concordance between bal gm and qpcr, even if bal cutoff for positivity of was applied (supplement fig. s ). there was no correlation between bal gm positivity and qpcr ct values and between pcr positivity and bal gm odi values (data not shown). the classification of ia in those receiving and not antifungal agents is outlined in table . the prevalence of positive and negative results of bal gm and aspergillus fumigatus qpcr in four different ia diagnostic categories, divided also into patients receiving and not mould active agents at the time of bal, is reported as supplement in table s . the performance of a. fumigatus qpcr is reported in table . the sensitivity was % ( %ci: - ) and specificity of % ( %ci: - ) when patients with proven/probable/possible ia were considered as cases, and, respectively, % ( %ci: - ) and % ( %ci: - ) considering as cases only those with proven/probable ia. sensitivity and specificity were similar also when patients with atypical ia were considered together with proven/probable ia as cases (table ) . when considering the influence of antifungal treatment, the sensitivity of qpcr was higher in those receiving mould active agents at the time of bal ( %, %ci: - ) compared to those not receiving antifungals ( %, %ci: - ), while the specificity was similar (respectively, %, %ci: - and %, %ci: - ) ( table ). among patients receiving mould active agents, nine had positive bal gm, and qpcr was positive in seven of them, compared to five among of those not receiving antifungals (p < . ). in all cases, the performance was much better when qpcr was used together with gm, and was worse if positivity of both qpcr and gm was required. the sensitivity values were similar irrespective of the definition used for cases (only those with proven/probable ia, those with proven/probable/possible ia, or those with proven/probable/atypical), being only slightly higher in case for proven/probable ia cases (table ) . if a cutoff for qpcr of ≤ ct was used, sensitivity was lower and specificity higher than for the cutoff of < ct. for proven/probable ia, the sensitivity was % ( %ci: - ) and the specificity was % ( %ci: - ), with higher sensitivity in those receiving mould active agents compared to those not in treatment, respectively, % ( %ci: - ) versus % ( %ci: - ). overall, out of patients with negative mycology and atypical lesions had a positive a. fumigatus qpcr ( %); eight of them received mould active drugs after bal (either as treatment or prophylaxis, including four patients who started mould-active agents before bal), while eight patients did not received any mould active agents within months after bal, and they were all alive at weeks follow-up. thus, considering high mortality of ia in patients with hematological malignancy if untreated, they were considered as false positives for qpcr or colonized. antifungal resistance tr /l h mutation was detected in one case, in a sct recipient previously exposed to azoles for prolonged treatment of ia after the first sct. after the second sct, gm in bal was positive but culture negative. initially after bal, azole therapy was started but after a clinical diagnosis of treatment failure, the therapy was changed to liposomal amphotericin b. gm became negative and radiological lesions improved, but the patient deceased months after the transplant due to a relapse of acute leukemia. this retrospective study showed a poor sensitivity of this qpcr for a. fumigatus, with better results in patients receiving mould active agents at the time of bal (sensitivity % vs %, respectively) and moderate specificity ( % and %, respectively). the combined performance of qpcr and gm was significantly better than the use of qpcr alone. our cohort included very selected patients, all at high risk for ia, with various radiological lesions, in whom bal was performed mainly because non-invasive results were negative for ia and diagnosis was not reached with other tests. moreover, % of them had lung lesions atypical according to eortc/msg criteria and positive mycological results. these patients are "unclassified" according to eortc, and they are usually considered as having ia and treated. therefore, they could not be included as controls for the assessment of diagnostic performance. however, even patients included in the control group (with non-typical lesions and negative mycology), belonged to a high risk population, and the positivity of pcr cannot be easily interpreted as false positive results. indeed, % of patients in this group with a positive pcr did receive empirically mould active agents, so only in remaining % of them, positive pcr could be confidently considered as false positive results or colonisation. moreover, even the presence of an alternative diagnosis should not serve a criterion for the absence of ia, since fungal and bacterial or viral co-infections are frequent in this setting. good diagnostic performances have reported in metaanalyses, which included mainly studies of in-house methods, with sensitivity and specificity reported of > % and > %, respectively. however, in some recent studies, the sensitivity was markedly lower (approximately %). , the commercial assays allow standardization, high reproducibility and have a validated quality control. however, they do not contain a control of dna extraction, which may differ for hyphae and for free dna, and quality control for the bal itself. additionally, their performance depends on the assay used and the clinical setting. the low value obtained in our study is not comparable with most of the performances described to date in the literature using mycogenie r . however, most other studies included patients with high rate of culture-positive respiratory samples. indeed, the sensitivity was . % in a cohort with of respiratory samples positive in culture for aspergillus, % in cases of probable ia with % of positive bal culture rate, and . % in case of fungal rhinosinusitis in which a high concentration of fungus dna is present. also a recent study comparing various diagnostic methods, which reported the sensitivity of mycogenie r of . % in patients with proven/probable ia, had a very high ( / , %) rate of positivity of culture for aspergillus. in addition, in that cohort, the sensitivity was lower in hematological patients than in those from intensive care unit (icu) suggesting a lower fungal burden sufficient to cause ifd in highly immunocompromised hosts. indeed, also the low yield of fungal cultures and rather low median bal gm odi confirm the low burden of viable moulds in this study. therefore, the performance of mycogenie r in a cohort of with low fungal burden remains to be established. another explanation for low sensitivity of this qpcr, even in a subgroup of bal gm positive patients with probable ia, might be the infection with species other than a. fumigatus which are not detected by this assay. a very interesting factor which influenced sensitivity in our cohort was the presence of mould active treatment at the time of bal, which increased the sensitivity from % to %. one possible explanation is that mould active agents caused lysis of the fungal wall with a higher percentage of free dna detectable in the respiratory tree, particularly in the supernatant of a centrifuged bal sample, which was the part used in this study. the availability of free fungal dna would result in much better sensitivity of dna extraction, which is a critical process for the performance of fungal pcr. this observation is indirectly confirmed by cohorts reporting higher performance of pcr, both in bal and serum, in those receiving mould active agents. [ ] [ ] [ ] also in our study, the rate of qpcr positivity in gm positive bal samples was higher form patients receiving mould active drugs compared to those not treated. better specification which portion of bal fluid should be used might be warranted, since by testing supernatant, dna still within the organism or phagocytic host cells might not be detected. the specificity of qpcr in our study was % which is lower than in the abovementioned meta-analyses and other studies using the same assay. , however, in this study there were no heathy controls, as all the patients were at high risk of ia and had lung lesions, which are referred to in a recent prospective eortc study as those in whom ia cannot be excluded. when considering the cases in which pcr represented the only positive mycological criterion, that is, with bdg and gm negative, half of these patients ( / ) survived > months without antifungal treatment and without developing ia, confirming that these were either false positives or cases of colonization. obviously, this could be established only due to a retrospective nature of our study in which pcr results were not available at the time of diagnosis, but it documents a high rate of clinically irrelevant false positive results. furthermore, considering the cross-reactivity with other species, in our study the qpcr, which should be specific for a. fumigatus, was positive in four cases in which different species grew in culture (two a. flavus, both with bal gm > . odi; one fusarium spp.; one paecilomyces). although in case of fungi other than aspergillus co-infection might be present, it is unlikely based on negative bal gm. on the contrary, cases of cross-reactivity of the method with strains of a. flavus have already been described in the literature, and such a false positive result might be particularly likely in case of high fungal burden, as demonstrated in our two cases. from the clinical point of view, a qpcr should detect all species of aspergilllus, since species other than a. fumigatus might be more prevalent, particularity in some geographical zones, for example, a. flavus in mediterranean. although triazole-resistant strains of a. fumigatus are increasing in several geographical regions, fortunately in our cohort only one patient had the tr /l h mutation, accounting for . % rate among pcr positive samples. such a low incidence is in line with what reported for italy; however, it should be considered with caution given overall poor sensitivity of this qpcr assay. although molecular methods are important tools, in addition to culture, to monitor the changes in resistance patters, also in this case, detection of more than one resistance mutation might be useful. finally, the absence of detected mutations does not exclude antifungal resistance in case of clinical failure since various mutation patterns can occur, particularly in case of previous azole exposure. the possibility of overestimating the performance of bal gm, which was the most frequent mycological criterion in this cohort, should be acknowledged. however, it could not be avoided in a population with mostly negative serum gm and bdg results, low culture yield, and lung biopsy frequently not feasible. in conclusion, our study demonstrated that the aspergillus fumigatus qpcr in bal should be performed together with gm, and it may offer clinical contribution particularly in patients receiving mould active agents, in whom gm is usually negative but pcr had better sensitivity. however, assays detecting most of the common species should be preferred and testing materials other than supernatant might result in higher sensitivity. pcr might be a valuable tool for detecting antifungal resistance both in case of infection or colonization, which may have significant implications for treatment and prophylaxis of ia. supplementary data are available at mmycol online. the clinical spectrum of pulmonary aspergillosis practice guidelines for the 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aspergillus pcr for testing serum and plasma: a study by the european aspergillus pcr initiative performance of molecular approaches for aspergillus detection and azole resistance surveillance in cystic fibrosis detection of aspergillus flavus and a. fumigatus in bronchoalveolar lavage specimens of hematopoietic stem cell transplants and hematological malignancies patients by real-time pcr, nested polymerase chain reaction and mmycological assays azole-resistant aspergillus fumigatus in the environment of northern italy financial support. this study was supported by the fellowship program award gilead . m.m. has received speaker and advisory board fees from gilead, pfizer, biotest, janssen and msd. c.v. has received research support to his institution from pfizer and msd, speaker, and advisory board fees from gilead, pfizer, and msd. other authors report no conflicts of interest. the authors alone are responsible for the content and the writing of the paper. key: cord- -b wm db authors: gaborit, benjamin jean; tessoulin, benoit; lavergne, rose-anne; morio, florent; sagan, christine; canet, emmanuel; lecomte, raphael; leturnier, paul; deschanvres, colin; khatchatourian, lydie; asseray, nathalie; garret, charlotte; vourch, michael; marest, delphine; raffi, françois; boutoille, david; reignier, jean title: outcome and prognostic factors of pneumocystis jirovecii pneumonia in immunocompromised adults: a prospective observational study date: - - journal: ann intensive care doi: . /s - - -x sha: doc_id: cord_uid: b wm db background: pneumocystis jirovecii pneumonia (pjp) remains a severe disease associated with high rates of invasive mechanical ventilation (mv) and mortality. the objectives of this study were to assess early risk factors for severe pjp and -day mortality, including the broncho-alveolar lavage fluid cytology profiles at diagnosis. methods: we prospectively enrolled all patients meeting pre-defined diagnostic criteria for pjp admitted at nantes university hospital, france, from january to january . diagnostic criteria for pjp were typical clinical features with microbiological confirmation of p. jirovecii cysts by direct examination or a positive specific quantitative real-time polymerase chain reaction (pcr) assay. severe pjp was defined as hypoxemic acute respiratory failure requiring high-flow nasal oxygen with at least % fio( ), non-invasive ventilation, or mv. results: of respiratory samples investigated during the study period, from patients were positive for p. jirovecii. of these patients, met criteria for pjp and were included in the study, ( . %) patients had severe pjp, including who required mv. all patients were immunocompromised with haematological malignancy ranking first (n = , %), followed by solid organ transplantation (n = , %), hiv-infection (n = , %), systemic diseases (n = , %), solid tumors (n = , %) and primary immunodeficiency (n = , %). by multivariate analysis, factors independently associated with severity were older age (or, . ; % ci . – . ; p < . ), a p. jirovecii microscopy-positive result from bronchoalveolar lavage (bal) (or, . ; % ci . – . ; p < . ); and absence of a bal fluid alveolitis profile (or, . ; % ci . – . ; p < . ). the -day mortality rate was %, increasing to % in the severe pjp group. factors independently associated with -day mortality were worse sofa score on day (or, . ; % ci . – . ; p < . ) whereas alveolitis at bal was protective (or, . ; % ci . – . ; p < . ). in the subgroup of hiv-negative patients, similar findings were obtained, then viral co-infection were independently associated with higher -day mortality (or, . ; % ci . – . ; p < . ). conclusions: older age and p. jirovecii oocysts at microscopic examination of bal were independently associated with severe pjp. both initial pjp severity as evaluated by the sofa score and viral co-infection predicted -day mortality. alveolitis at bal examination was associated with less severe pjp. the pathophysiological mechanism underlying this observation deserves further investigation. over the last years, survival benefits provided by steady advances in antitumor chemotherapy and immunosuppressant regimens for patients with autoimmune diseases, haematological malignancies, and solid organ transplants have substantially increased the number of adults living with immunodeficiencies [ , ] . among opportunistic infections in immunocompromised adults, pneumocystis jirovecii pneumonia (pjp) was associated with high rates of intubation and mortality [ ] . consequently, an early identification and optimal treatment of patients with pjp remains a key priority [ , ] . since the advent of antiretroviral therapy, the incidence and mortality rates of pjp among patients positive for the human immunodeficiency virus (hiv) have decreased steadily [ ] . however, pjp is being increasingly diagnosed in hiv-negative patients, in whom it carries a poorer prognosis [ , ] . a higher proportion of neutrophils in broncho-alveolar lavage (bal) fluid during pjp was associated with higher risks of respiratory complications and mortality [ , ] . in the same way, a low lymphocyte count in bal fluid was a risk factor for the failure of trimethoprim/sulfamethoxazole (tmp/smz) therapy [ ] . early adjunctive steroid therapy for severe pjp dramatically decreased mortality rates in hiv-positive patients [ , ] but had variable effects in their hivnegative counterparts [ ] [ ] [ ] . these findings suggest that the immunological status and underlying diagnosis may influence the pathophysiology of pjp and the risk of mortality [ , ] . however, bal fluid cytology profiles have not been adequately evaluated as potential prognostic factor and predictors of treatment responses. the identification of early predictors of pjp outcomes, including bal fluid findings, may help to determine which patients are most likely to benefit from intensive care and could justify adjunctive steroid therapy. the aim of this prospective study of patients with pjp was to identify early risk factors for severe pjp and -day mortality. this is a retrospective analysis of prospective cohort. from january to january , all patients presenting an invasive fungal infection that has been diagnosed in our center have been included in a prospective registry (the french prospective surveillance programme, ressif network), thus pjp patients have been included in a subcohort. for each patient with a positive sample, the following clinical findings were prospectively investigated: dyspnea and/or cough in immunocompromised patients with interstitial syndrome by radiography or ct scan. among all respiratory samples investigated for years (n = ), all positive tests for pneumocystis jirovecii samples (n = ) were assessed by a biologist and a clinician to investigate criteria for pjp and include them in this prospective cohort. the following data were prospectively collected: age; sex; underlying disease; pjp prophylaxis; other medications including glucocorticoids taken during the past month; type of symptoms and symptom duration at pjp diagnosis and time from symptom onset to hospital admission. secondary, the clinical data were collected for all patients from medical records: laboratory findings (white blood cell count; absolute neutrophil count; c-reactive protein [crp] level; bal fluid findings including the cell profile assessed by an independent cytologist on centrifuged bal fluid samples prepared with the wright-giemsa and perls stains to allow the determination of macrophage, lymphocyte, neutrophil, eosinophil, and basophil counts); presence of p. jirovecii and/or other fungi and/or bacteria and/or viruses (influenza viruses, respiratory syncytial virus, adenovirus, cytomegalovirus) were recorded at pjp diagnosis. the sofa score on day and the ratio of the arterial partial pressure of oxygen over the fraction of inspired oxygen (pao /fio ) on day [ ] were recorded for each patient at pjp diagnosis. anti-pjp medications, adjuvant glucocorticoid therapy, oxygen supplementation, and ventilatory support provided at admission were documented. finally, patient outcomes including -day mortality were recorded. the collection of follow-up data ended in december . severe pjp was defined as hypoxemic acute respiratory failure requiring high-flow nasal oxygen with at least % fio , non-invasive ventilation, or mv. because not all patients were hospitalized in intensive care units, we chose this pragmatic and reproducible severity criterion. according to the berlin definition [ ] , severe acute respiratory distress syndrome (ards) was defined as the presence of the following criteria within days after icu admission: new respiratory symptoms, bilateral opacities mortality. alveolitis at bal examination was associated with less severe pjp. the pathophysiological mechanism underlying this observation deserves further investigation. keywords: pneumocystis jirovecii pneumonia, early prognostic score, high flow oxygen, haematological malignancies, alveolitis on chest radiographs or by ct, absence of suspected hydrostatic/cardiogenic pulmonary oedema, and pao / fio ≤ . lymphocytic alveolitis [ ] was defined as a bal fluid cell population containing more than % of lymphocytes and more than % of neutrophils combined with an activated macrophage phenotype, based on the diagnostic criteria for hypersensitivity pneumonitis, a condition characterised by alveolitis and migration to the alveoli of multiple cell types including activated t cells, monocytes, and natural killer cells [ ] . finally, patients with other pathogens associated with p. jirovecii in respiratory or blood samples were classified as having coinfection at icu admission. patient characteristics were described using mean ± sd for continuous variables (or % confidence interval [ % ci] when appropriate) and proportions for qualitative variables. continuous variables were compared using the wilcoxon rank-sum and kruskal-wallis tests and qualitative variables using fisher's exact test with computation of the odds ratios (ors) and their % cis. overall survival was assessed using kaplan-meier curves and log-rank tests. factors associated with severity were identified by logistic regression analysis. factors associated with all-cause -day mortality were identified by logistic regression analysis of patients alive after day versus patients who died before day . only factors reaching statistical significance (p < . ), with no more than % missing data, were included in the multivariate model. when collinearity between co-variates was detected, only the variable with the highest or was kept into the multivariate regression model. results are reported as log-transformed coefficients with their % cis. missing data were not interpolated. analyses were performed on the whole population, then excluding hiv+ patients. of the respiratory samples tested during the study period, from patients were positive for p. jirovecii. of these patients, met our pjp criteria and were included in the study; table reports their main characteristics. the remaining patients were classified as having bronchial p. jirovecii colonisation (fig. ). the mean number of patients included per year was and the number of patients per year increased gradually over time to reach a peak of patients in (additional file : figure s ). of the patients with pjp, had positive bal fluid, by a direct examination of bal fluid in patients and only detected by pcr in patients. the induced sputum test was positive in patients (by pcr, n = ; time from onset to bal, days, mean ± sd ± . pj visible in smears, n (%) ( . ) alveolitis profile, n (%) ( ) co-infection at pjp diagnosis, n (%) viral infection ( ) bacterial infection ( ) invasive fungal infection ( ) and/or grocott-gomori stain, n = ). both the bal fluid and the induced sputum test were positive in patients. all patients were immunocompromised with haematological malignancy ranking first (n = , %), followed by solid organ transplantation (n = , %), hiv-infection (n = , %), systemic diseases (n = , %), solid tumors (n = , %) and primary immunodeficiency (n = , %). the mean hiv viral load at pjp diagnosis was , copies for hiv-positive patient. only ( . %) patients were given pjp prophylaxis. of these, were compliant with the prescription during the last months before diagnosis, which never consisted in tmp/smz. the first-line pjp therapy was tmp/smz for ( . %) patients, ( . %) patients being treated with atovaquone; no pjp therapy was given to the remaining patient, who died within h after icu admission (pjp diagnosis was made post-mortem). adjunctive glucocorticoid therapy was given to ( %) patients based on severity criteria. sixteen patients were switched from tmp-smz to atovaquone (n = ) or pentamidine (n = ), due to acute kidney injury (n = ), myelotoxicity (n = ) allergy (n = ), or hepatic cytolysis (n = ); of these patients had both acute kidney injury and myelotoxicity. of patients with available bal fluid cytology results, ( %) had evident alveolitis profile. mean bal fluid percentages in the patients were . ± . for neutrophils, ± . for macrophages, and ± . for lymphocytes. bacterial co-infection was diagnosed in ( %) patients, viral co-infection in ( %) patients, and fungal co-infection in ( %) patients (additional file : table s ). lymphocytes and neutrophils mean ratio were, respectively, % and % in hiv patients, % and % in non-hiv patients, % and % in dead patients, % and % in survivors patients, % and % with only pjp patients, % and % during coinfection. we did not observe in our study eosinophilic and neutrophilic alveolitis. of the patients, ( . %) were classified as severe pjp (table ) . icu admission was required in patients, including who received mv. the hiv serology was positive in / ( %) patients with severe pjp and / ( %) patients with non-severe pjp. early risk factors associated with severity in the univariate analyses were age > years (or, . ; % ci . - . ; p < . ), albuminemia < g/l (or, . ; % ci . - ; p < . ), blood neutrophil count > . g/l (or, . ; % ci . - ; p < . ), bal fluid neutrophils > % (or, . ; % ci - ; p < . ), p. jirovecii oocysts observed at direct examination of bal fluid (or, . ; % ci . - . ; p < . ), higher baseline lactic dehydrogenase value (or, . ; % ci . - . ; p < . ), and higher crp (or, . ; % ci . - . ; p < . ). a bal alveolitis profile was protective (or, . ; % ci . - . ; p < . ). by multivariate analysis, factors independently associated with severe pjp were older age (or, . ; % ci . - . ; p < . ), p. jirovecii oocysts observed at direct examination of bal fluid (or, . ; % ci . - . ; p < . ) and the absence of a bal fluid alveolitis profile (or, . ; % ci . - . ; p < . ). bmi, body mass index; icu, intensive care unit; ards, acute respiratory distress syndrome; saps , simplified acute physiology score version ; sofa score, sequential organ failure assessment score; hiv, human immunodeficiency virus; pjp, pneumocystis jirovecii pneumonia; crp, c-reactive protein; ldh, lactate dehydrogenase; pj, pneumocystis jirovecii a the total exceeds % because some patients had more than one cause of immunodeficiency b of these patients, followed their prescribed prophylactic regimen (aerosolised pentamidine, n = ; and atovaquone, n = ) and did not (trimethoprim/sulfamethoxazole, n = ; and aerosolised pentamidine, n = ) two factors were independently associated with -day mortality by multivariate analysis, a worse sofa score was associated with higher -day mortality (or, . ; % ci . - . ; p < . ), whereas bal fluid alveolitis profile was associated with lower -day mortality (or, . ; % ci . - . ; p < . ) ( table ) . hiv serology was a protective factor in univariate analysis but was not statistically associated with protective factor in the multivariate -day mortality analysis. in survival analysis hiv patients presenting with pjp was associated with statistically better prognostic than that of patients with hematologic diseases or solid cancer (additional file : figures s , s ) . in the subgroup of hiv-negative patients, similar findings were obtained, then viral co-infection were independently associated with higher -day mortality (or, . ; % ci . - . ; p < . ) (additional file : tables s , s ). factors associated with -day mortality in icu patients were sofa score and non-hiv patients (additional file : table s ). in this prospective study, over four-fifths of pjp patients were hiv-negative, and half met our criteria for severe disease. a worse sofa score on admission and viral co-infection were independently associated with higher -day mortality in both whole patients and hiv-negative patients. importantly, a bal fluid cytological profile consistent with alveolitis was associated with lower -day mortality. severe pjp on admission as defined for our study was associated with a % risk of receiving mv, in keeping with recent results from large cohort studies [ , ] . given the prognostic significance of severity, an improved knowledge of early risk factors for severity is helpful to identify patients requiring more intensive monitoring and treatment. as illustrated here, hiv patients less often experienced severe pjp compared to their hiv-negative counterparts, in agreement with earlier data [ , , [ ] [ ] [ ] [ ] . the reduced severity of pjp in hiv-positive patients is more table risk factors for -day mortality in the overall population (patients where bal was performed, n = ) italics characters correspond to the analysis parameters with a statistically significant difference bmi, body mass index; saps , simplified acute physiology score version , sofa score, sequential organ failure assessment score; hiv, human immunodeficiency virus likely to be associated with the particularity of hivinduced immunosuppression, of which pneumocystis is a hallmark of a severe adaptive cellular impairment. the co-morbidities in non-hiv patients (onco-hematology, solid organ transplantation and system diseases) may also contribute to their greater vulnerability. the immune recovery allowed by the initiation of antiretroviral therapy is probably correlated with better long-term outcomes in hiv-positive patients than in non-hiv patients whose profound immunosuppression is more frequently extended. hiv-positive patients also have lower neutrophil counts in bal fluid samples [ ] , are less likely to develop severe pjp, and have lower mortality rates compared to hiv-negative patients [ , [ ] [ ] [ ] [ ] . the sofa score, viral co-infection and absence of alveolitis remained independently associated with a -day higher mortality in hiv-negative patients, suggesting that it is a strong independent marker in the whole pjp population. by univariate analysis, early risk factors for severity in hiv-negative patients were markers for vulnerability (older age and lower serum albumin) and for inflammation (systemic inflammatory syndrome characterized by blood and alveolar polynucleosis serum crp level). the patient subgroup at the most severe end of the spectrum had the highest crp levels and neutrophil counts, suggesting that anti-inflammatory treatments might improve patient outcomes. our study suggests that future prospective studies on adjunctive treatments for pjp should focus on hiv-negative patients, notably solid organ and haematological stem cell transplant recipients, meeting criteria for severe pjp (e.g., sofa score > with a low pao /fio ratio). the potential relevance of a bal fluid cytology profile consistent with alveolitis should probably also be taken into account when assessing the efficacy of new treatment regimens. as expected, mortality within the first days was significantly higher in patients with severe versus nonsevere pjp. the independent association between a worse sofa score at admission and -day mortality confirms the appropriateness of an evaluation with an intensivist to consider icu admission of patients with oxygen-dependent pjp [ ] . the quicksofa, which is a simplified score based on three criteria, is easy to determine by non-intensivists and may be useful for determining when advice from an intensivist should be sought [ ] . in addition to a worse sofa score, viral co-infection at the time of pjp diagnosis was associated with higher -day mortality. viral infections consisted mostly ( %) in reactivation of latent viruses (cytomegalovirus, herpes simplex virus, or epstein-barr virus), suggesting that this subgroup may have been characterised by a more profound immune deficiency. taken together, this finding supports the hypothesis that the outcome of pjp is also closely related to the underlying diagnosis and immune response. an important finding from this study is the clear association between a bal fluid cytology profile consistent with alveolitis (> % lymphocytes, > % neutrophils, and presence of activated macrophages) and less severe pjp and lower -day mortality. the presence of neutrophils in bal fluid has often been noted in clinical and experimental studies of acute respiratory distress syndrome (ards) [ , ] . three patient subgroups can be identified in our population, one defined by alveolitis, the other one pjp occuring in hiv-positive individuals, both having a better prognosis and the last one defined by a sofa score above on admission who have a more severe prognosis. taken together, these results suggest that specific immunological characteristics might allow the identification of patient subgroups with different treatment needs and outcomes. cd + t cell levels were non-significantly higher in patients with alveolitis, whereas glucocorticoid exposure was comparable in the two groups. to our knowledge, alveolitis during pjp has not been described as a good prognostic factor yet [ ] . in murine models of cd + t-cell depleted mice, increased alveolar recruitment of cd + t cells induced by interleukin- therapy improved p. jirovecii clearance [ ] . lymphocyte count in bal fluid is increased in patients with alveolitis, which was associated with a better prognosis in our study. thus, alveolitis during pjp might reflect maintenance of an effective pulmonary immune response including cd + t-cell recruitment. glucocorticoid therapy might, therefore, be unnecessary in patients with pjp and alveolitis, although their specific response to glucocorticoids has not been investigated to date. four-fifths of our patients had not been prescribed pjp prophylaxis, and among those with a prescription only one-third were compliant. these findings confirm earlier results [ , ] and further identify the absence of tmp/smz prophylaxis as a major risk factor for pjp in high-risk patients [ ] . providing appropriate prophylactic anti-microbial treatments to patients with immunosuppression, notably related to haematological diseases and transplantation, is crucial to improve patient outcomes. adherence to prophylactic treatment must be supported at each follow-up visit. pjp usually develops in patients with cd + counts below /mm [ ] [ ] [ ] , although the depth of lymphopenia does not correlate with pjp severity [ , ] . a history of glucocorticoid exposure is an often reported risk factor for pjp in hiv-negative patients [ , ] . among our patients, over half were receiving glucocorticoid therapy at the diagnosis of pjp, in a mean prednisone-equivalent dosage of mg/day. adjuvant glucocorticoid therapy was given to most severe patients who failed antibiotic treatment alone. given the history of glucocorticoid exposure, the effects of adjuvant glucocorticoid therapy were difficult to assess. the role for adjuvant glucocorticoid therapy in patients with pjp is debated [ , [ ] [ ] [ ] [ ] [ ] and is currently being assessed in a prospective study (clinicaltrials.gov identifier: nct ). serum ldh level at pjp diagnosis was associated with poor outcomes in hiv-positive and hiv-negative patients 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of pneumocystis jirovecii pneumonia in cancer patients analysis of underlying diseases and prognosis factors associated with pneumocystis carinii pneumonia in immunocompromised hiv-negative patients clinical course, treatment and outcome of pneumocystis pneumonia in immunocompromised adults: a retrospective analysis over years pneumocystis carinii infection: current treatment and prevention publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the clinicians and microbiologists who contributed to the management of the study patients for their commitment to providing optimal patient care. supplementary information accompanies this paper at https ://doi. org/ . /s - - -x. abbreviations ards: severe acute respiratory distress syndrome; bal: broncho-alveolar lavage; bmi: body mass index; ci: confidence interval; cmv: cytomegalovirus; crp: c-reactive protein; ebv: epstein-barr virus; fio : fraction of inspired oxygen; hiv: human immunodeficiency virus; hsv : herpes simplex virus; icu: intensive care unit; ldh: lactate dehydrogenase; mv: invasive mechanical ventilation; niv: non-invasive ventilation; or: odds ratio; pao : arterial partial pressure of oxygen; pcr: real-time polymerase chain reaction; pjp: pneumocystis jirovecii pneumonia; rsv: respiratory syncytial virus; saps : simplified acute physiology score version ; sd: standard deviation; sofa score: sequential organ failure assessment score; tmp-smx: trimethoprim-sulfamethoxazole.authors' contributions bjg and bt analysed and interpreted the data. cs performed the cytological examinations of broncho-alveolar fluid samples. fm, ral, and bjg were members of the independent committee that adjudicated the pjp cases. bjg and jr wrote the manuscript. all authors read and approved the final manuscript no funding was received for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. all authors read the final version of the manuscript and approved its submission to annals of intensive care. the authors declare that they have no competing interests. key: cord- -xdic rcy authors: petini, matteo; furlanello, tommaso; danesi, patrizia; zoia, andrea title: nested–polymerase chain reaction detection of pneumocystis carinii f. sp. canis in a suspected immunocompromised cavalier king charles spaniel with multiple infections date: - - journal: sage open med case rep doi: . / x sha: doc_id: cord_uid: xdic rcy a -month-old cavalier king charles spaniel female was referred due to a chronic cough refractory to antibiotic treatments. laboratory findings showed leukocytosis, increased serum c-reactive protein, hypogammaglobulinemia, and decreased total serum immunoglobulin g concentration. thoracic radiographs showed a mild bronchial pattern. cytology of the bronchoalveolar lavage fluid revealed a septic inflammation. bordetella bronchiseptica, mycoplasma spp., and pneumocystis carinii were identified by polymerase chain reaction testing, and klebsiella pneumonia was cultured from the bronchoalveolar lavage fluid. moreover, escherichia coli was also cultured from urine. pneumocystis spp. identification was done by sequencing of genetic amplicons. the dog died due to cardiopulmonary arrest secondary to a spontaneous pneumothorax on the day following the procedure. this report documents the detection of pneumocystis carinii f. sp. canis in a suspected immunocompromised cavalier king charles spaniel with concurrent pulmonary and urinary tract infections involving four different pathogens, and highlights the importance of the use of polymerase chain reaction testing to detect canine pneumocystis spp. in cases with negative bronchoalveolar lavage cytology. the genus pneumocystis contains highly diversified and opportunistic fungal species that cause severe pneumonia in mammals with a deficient immune system. in the veterinary literature, there are many published cases of confirmed canine pneumocystosis-most of which described cases of young to middle-age dogs with suspected immunodeficiency, with the miniature dachshund, and the cavalier king charles spaniel (ckcs) breeds most commonly reported. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] concurrent infection with other pathogens has been reported in only a few cases when concurrent demodex canis , , and canine distemper virus (cdv) infections were demonstrated. in most of the previously reported veterinary cases, in vivo diagnosis of pneumocystis spp. pneumonia (pp) was achieved by direct identification of the microorganism by cytological examination of bronchoalveolar lavage (bal) samples, trans-tracheal aspirates, or transthoracic lung aspirates. however, using this technique, the diagnosis can be missed due to failure on detection of the lung pathogen. , , , in human medicine, several studies have demonstrated that polymerase chain reaction (pcr)-based methods increase the sensitivity of detection of the organism compared to its direct identification on lung samples. , indeed, real-time pcr has been recently recommended for pp diagnosis by a european panel of experts. the aim of this report was to describe the first case of pneumocystis carinii f. sp. canis infection in a suspected immunocompromised ckcs, with four concurrent pulmonary infective agents and one extrapulmonary infectious agent. in this case, an in vivo identification of pneumocystis spp. was achieved by pcr testing of bal fluid, in which no pneumocystis cysts had been identified with cytological examination. a -month-old ckcs female, with a history of chronic coughing, was referred to the "san marco veterinary clinic" (padova, italy) for further investigations. the dog was not vaccinated and had not received any heartworm prophylaxis in the previous months. consecutive antibiotic treatments with appropriate doses of doxycycline, metronidazole-spiramycin, and finally, a -day course of amoxicillin-clavulanic acid all had all failed to resolve or even improve clinical signs. antibiotic treatment was discontinued days before presentation. two previous fecal baermann's tests were negative for angiostrongylus vasorum. pcr testing of a nasal swab for cdv, adenovirus (cav- ), herpesvirus , influenza virus, parainfluenza virus (cpiv), and respiratory coronavirus was also negative. on presentation, the dog was bright and alert, mildly tachypneic ( r/min) with a bilateral mucous nasal discharge. the remaining physical examination was normal. a blood sample was taken for a complete blood count, including blood smear examination, serum biochemistry, and serum electrophoresis, and for a coagulation profile. urinalysis was performed on a urine sample obtained via cystocentesis. right lateral and dorso-ventral thoracic radiographs were also taken. clinicopathological abnormal findings included a mild nonregenerative, normocytic normochromic anemia ( . × /l; reference interval, . - . ), leukocytosis ( . × /l; reference interval, . - . ) with neutrophilia ( . × /l; reference interval, . - . ), lymphocytosis ( . × /l; reference interval . - . ), and monocytosis ( . × /l; reference interval, . - . ). on blood smear examination, there were activated lymphocytes and toxic neutrophils. serum biochemical profiles showed an increase in serum c-reactive protein ( . mg/l; reference interval, . - . ), a mild hypoalbuminemia ( g/l; reference interval, - ), and a mild hypoglobulinemia ( g/l; reference interval, - ). serum immunoglobulin fraction quantification showed a decreased immunoglobulin g (igg) concentration ( . g/l; reference interval . - . ), while immunoglobulin m (igm) and immunoglobulin a (iga) concentrations were at the lower limit of the reference intervals ( . g/l; reference interval, . - . and . g/l; reference interval, . - . ; respectively). serum globulin quantification was in agreement with those detected by micro-capillary electrophoresis diagram (table and figure ). rod-shaped bacteria and pyuria were detected with urine sediment examination and therefore urine culture was performed. all the remaining clinicopathological tests were normal. the radiographs of the thorax showed a mild diffuse bronchial pattern (figure rhinoscopy, bronchoscopy, and bal were performed to further elucidate the clinical signs. the dog was pre-medicated with butorphanol ( . mg/kg, im) and dexmedetomidine ( . mg/kg, im), induced with propofol ( mg/kg) and maintained using a propofol infusion ( . - . mg/kg/ min). flow by % oxygen delivery was provided during the entire procedure until the dog was fully recovered. a small flexible fiberoptic bronchoscope was used. pulse oximetry, electrocardiography (ecg), and blood pressure were monitored throughout the procedure. a diffuse, productive bronchopathy and a non-specific mucous-productive rhinopathy were identified. bal was performed by instilling two aliquots of ml of warmed sterile . % saline by syringe, followed by approximately ml of air to clear the fluid from the endoscope channel before the saline was aspirated. the bal sample was immediately submitted for cytological analysis and bacterial culture. polymerase chain reaction testing for cdv, cav- , cpiv, angiostrongylus vasorum, bordetella bronchiseptica, mycoplasma spp., and pneumocystis spp. was also performed. to investigate the presence of pneumocystis spp., a portion of the mitochondrial small subunit rrna gene (mtssu rrna gene) was amplified and sequenced by nested pcr with previously reported primers and protocol. cytological examination of the bal fluid revealed a suppurative inflammation associated with the presence of intracellular coccobacilli bacteria. urine culture and bal fluid culture were positive for escherichia coli ( , , cfu) and klebsiella pneumonia, respectively. both microorganisms were sensitive to all antibiotic molecules tested on the antibiogram, and, surprisingly, included those used up to days before presentation (i.e. amoxicillin-clavulanic acid). polymerase chain reaction testing of bal fluid was positive for b. bronchiseptica, mycoplasma spp., and pneumocystis spp. pneumocystis identity was confirmed by sequencing of the mtssu rrna amplicon. the unroot phylogenetic tree constructed using mtssu rrna sequences showed that pneumocystis carinii f. sp. canis was distinct from pneumocystis spp. isolated from other animals, confirming that this was the specific species related to canine hosts (figure ). at the owner's request, at the end of all diagnostic procedures, the dog was discharged from the hospital, pending results and against medical advice. at this time, the dog had fully recovered from the anesthesia and physical examination was unchanged compared to the morning admission. on the following morning, the dog returned in acute respiratory distress. thoracic radiographs showed a severe pneumothorax, and the animal died a short time later from cardiopulmonary arrest despite attempts to stabilize it. post-mortem examination was declined from the owner. this case report, in the authors' opinion, highlights four points about the diagnosis and clinical presentation of dogs with pneumocystis spp. first, in our laboratory, all reference intervals are calculated using a population of dogs matched for age, breed, and sex to the patient being tested. dogs with chronic inflammatory disease (such as chronic septic pneumonia as in this case) often present with hypergammaglobulinemia and normal-to-high igg concentration. the decreased gammaglobulins and igg concentrations identified in this patient, together with the igm and iga in the lower limits of the reference intervals, were highly suspicious of an immunocompromised state, in agreement with previously reported cases of pp in ckcs. , alternatively, the lack of vaccinations in this dog may has contributed to the low serum concentrations of gammaglobulins and igg concentrations, since reference intervals are derived from vaccinated animals. second, the detection of the fungal pathogen was achieved through pcr testing of a bal sample which was negative with microscopic visualization for pneumocystis spp. it must be considered that all pneumocystis spp. have two developmental stages: the trophic form (formerly trophozoite) and the cyst form. the imbalance between the different lifecycle forms of pneumocystis spp. might explain the lower diagnostic yield of cytology in certain instances, as cysts are much easier to distinguish than trophic forms using rapid, modified romanowsky stains such as diff-quik. such an explanation might account for why some specimens can be pcr-positive, yet no organisms can be detected using conventional light microscopy. , , , similar to humans, pcr method to achieve a pp diagnosis has been suggested in dogs, , but it has only recently been used ante-mortem. , these recent studies confirm that pcr testing can be a valid diagnostic test for the detection of pneumocystis spp. in addition, the phylogenetic analysis of the pneumocystis spp. nucleic acid amplified from bal fluid in the current study showed that it was a specific canine host-related species. the third peculiarity of this case report was the multiple bacteria identified by pcr and cultured from the bal specimen and the presence of a concurrent urinary bacterial infection (i.e. e. coli). although pulmonary co-infection with b. bronchiseptica has previously been reported, multiple coinfections with k. pneumonia, mycoplasma spp., and bordetella bronchiseptica, contemporary to a urinary tract infection with e. coli, have never been described before, further supporting the immune-suppression state of the dog. moreover, it is also important to underline that all bacterial agents isolated by pcr and cultured are usually sensitive to one or more of the antimicrobial agents previously administered to the dog, supporting further the presence of an immunodeficient state in this patient. the final unusual finding of this report was that despite the detection of four different potential respiratory pathogens from the bal sample, the lung changes in the thoracic radiographs were not especially severe, nor typical of pp. , a possible explanation for this finding could be that the patient was in an initial stage of pp. alternatively, it is possible that the pneumocystis carinii f. sp. canis detected by pcr was just a colonizing respiratory tract of the dog without playing an active part in the pneumonia. finally, the recent and multiple antibiotic treatments may have mitigated the intensity of the radiographic changes. in veterinary literature regarding pp, pneumothorax it has been reported only following a lung fine needle aspiration on a ckcs. however, in human medicine, it is described as a clinical finding in %- % of human patients affect by pp. lung histology in these patients revealed a significant peripheral lung destruction characterized by sub-pleural necrosis and the presence of small and large sub-pleural cystic spaces. in this study, the patient's pneumothorax may have occurred secondary to airway endoscopy and bal. finally, the lung damage caused by multiple infectious agents may also had played a role in the pathogenesis of the pneumothorax. unfortunately, at the owner's request and against medical advice, the dog was discharged from the hospital soon after the bal procedure, preventing a watchful observation in an intensive care environment which may had altered the final outcome. one limitation of this case report is that we were not able to establish the fungal load and the pathological impact of pneumocystis spp. on the respiratory signs of this dog. quantitative pcr assay to estimate the fungal load would have been useful for this purpose. unfortunately, this technique was not available at the time of presentation of the dog reported here. while signalment, medical history, clinicopathological, and nested pcr findings supported a pp diagnosis in our dog, the radiographic findings were not consistent with pp. however, in human medicine, it is strongly recommended to consider the detection of pneumocystis spp. dna in symptomatic patients, whatever the fungal load, to be at least partially, responsible for the clinical signs. pneumocystosis in dogs: metaanalysis of published cases including clinical signs, diagnostic procedures, and treatment pneumocystis carinii pneumonia in dogs-a diagnostic challenge occurrence of pneumocystis carinii in canine distemper pneumocystis carinii pneumonia in two cavalier king charles spaniels common variable immunodeficiency in miniature dachshunds affected with pneumonocystis carinii pneumonia pneumocystis pneumonia in two cavalier king charles spaniel littermates common variable immune deficiency in a pomeranian with pneumocystis carinii pneumonia pneumocystis carinii infection with severe pneumomediastinum and lymph node involvement in a whippet mixedbreed dog molecular diagnosis of pneumocystis pneumonia in dogs improved detection of pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time pcr: a prospective study pneumocystis pcr: it is time to make pcr the test of choice ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients barcoding markers for pneumocystis species in wildlife immunologic and plasma protein disorders pneumocystis carinii pneumonia in a cavalier king charles spaniel immunoglobulin deficiency in cavalier king charles spaniels with pneumocystis pneumonia immune modulation following immunization with polyvalent vaccines in dogs radiographic aspects of pneumocystis carinii pneumonia in the miniature dachshund recurrent pneumothorax in aids patients with pneumocystis pneumonia: a clinicopathologic report of three cases and review of the literature (eds) textbook of veterinary internal medicine: disease of the dog and cat pneumocystis jivorecii detection in asymptomatic patients: what does its natural history tell us? the authors thank dr mark westman for editing the manuscript. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. all the procedures performed complied with the european legislation (directive / /eu) and with the ethical requirement of the italian law (decreto legislativo / / , n. ). accordingly, this type of study does not require an authorization or an id protocol number. the author(s) received no financial support for the research, authorship, and/or publication of this article. informed written consent was obtained from the dog's owner. matteo petini https://orcid.org/ - - - key: cord- -ekwpkzqv authors: bewig, b.; stewart, susan; böttcher, heidi; bastian, andreas; tiroke, andreas; hirt, stefan; haverich, axel title: eosinophilic alveolitis in bal after lung transplantation date: journal: transpl int doi: . /s sha: doc_id: cord_uid: ekwpkzqv lung transplantation has become a therapeutic option for patients with end stage lung disease. however, outcome after transplantation is complicated by episodes of rejection and infections. bronchoalveolar lavage is a valuable tool in monitoring patients after transplantation, since it allows the detection of pathogens. a marker specifically indicating rejection from changes in bal fluid has not been found yet. especially changes in differential cell count, like lymphocytosis or an increase in polymorphnuclear granulocytes, are unspecific. the role of high eosinophil levels in bal has not been elucidated yet. we analyzed bal samples and clinical data of patients who underwent lung transplantation and presented with recurrent episodes of eosinophilic alveolitis in bal. all patients demonstrated a deterioration of clinical condition, lung function, and blood gas analysis during times of eosinophilia in bal, compared to previous examinations. in all cases, eosinophilia in bal was accompanied by rejection. all patients were finally treated with high doses of steroids, resulting in improvement of all parameters. eosinophilia was not associated with significant changes in the il- concentration in bal or the pattern of il- expression in bal cells. in conclusion, eosinophilic alveolitis may indicate acute rejection in patients after lung transplantation, if other causes of eosinophilia are excluded. abstract lung transplantation has become a therapeutic option for patients with end stage lung disease. however, outcome after transplantation is complicated by episodes of rejection and infections. bronchoalveolar lavage is a valuable tool in monitoring patients after transplantation, since it allows the detection of pathogens. a marker specifically indicating rejection from changes in bal fluid has not been found yet. especially changes in differential cell count, like lymphocytosis or an increase in polymorphnuclear granulocytes, are unspecific. the role of high eosinophil levels in bal has not been elucidated yet. we analyzed bal samples and clinical data of patients who underwent lung transplantation and presented with recurrent episodes of eosinophilic alveolitis in bal. all patients demonstrated a deterioration of clinical condition, lung function, and blood gas analysis during times of eosinophilia in bal, compared to previous examinations. in all cases, eosinophilia in bal was accompanied by rejection. all patients were finally treated with high doses of steroids, resulting in improvement of all parameters. eosinophilia was not associated with significant changes in the il- concentration in bal or the pattern of il- expression in bal cells. in conclusion, eosinophilic alveolitis may indicate acute rejection in patients after lung transplantation, if other causes of eosinophilia are excluded. lance of patients after lung transplantation. in this situation, careful analysis of cytological, microbiological, and clinical data becomes all the more important for the correct diagnosis and treatment of complications after lung transplantation. we report data demonstrating that some patients present with eosinophilic alveolitis in bronchoalveolar lavage fluid early after transplantation. after exclusion of infections typically causing eosinophilic alveolitis, such as aspergillus, this finding may be indicative for acute rejection episodes. in this retrospective study, clinical data and differential cell counts from bal samples of lung transplant recipients, who had been treated at the university of kiel until december and were available for follow-up investigation, were analyzed. of the recipients, received a single lung, a bilateral-, and a heartlung transplant. for indications of lung transplantation see table . observation time was at least months. a total of bal samples was screened for eosinophilia. patients developing eosinophilic alveolitis were selected for further retrospective analysis. immunosuppression was started intraoperatively with a single dose of g methylprednisolone (urbason Ò , hoechst, bad soden) i. v., followed by days of mg and a month oral taper from . mg/ kg to a dose of . mg/kg per day methylprednisolone. all patients received mg/kg body weight azathioprine (imurek Ò , glaxo wellcome, hamburg) intraoperatively. beginning on day after transplantation, azathioprine was given at a dose of mg/kg, adjusted to maintain blood neutrophils just above /ml. immediately after transplantation, administration of cyclosporine a (sandimmun Ò , sandoz, nürnberg) was initiated maintaining a blood level of ± mg/l. therapy for rejection episodes consisted in methylprednisolone pulse therapy ( . ± g/day i. v. for days). in some cases of ongoing or recurrent rejection, cytolytic therapy with rabbit-antithymocyte globuline (tecelac Ò , biotest, dreieichen) was administered for days at . ± . mg/day, followed by a steroid taper from . to . mg/kg, which was applied for weeks. bronchoscopy and bal were performed weekly during the first months, every weeks for the next months, and at months , and after transplantation. additional bal samples were obtained if patients showed clinical, lung-functional, or radiographic deterioration. bronchoscopy was carried out using flexible fiberoptic bronchoscopes (olympus, hamburg). patients received atropine . mg (atropinsulfat Ò , braun, melsungen) and hydrocodon . mg s. c. (dicodid Ò , knoll, ludwigshafen) half an hour before examination. up to ml % oxybuprocainhydrochloride (novesine Ò ,wander, nürnberg) or % lidocainehydrochloride (xylocain Ò , astra, hamburg) were used as local anaesthesia. supplemental oxygen was administered at a rate of ± l/min. bal was performed in the middle lobe bronchus or in the bronchial segment affected by infiltrates. ml of . % sodium chloride solution were applied in ml fractions, each fraction being aspirated by gentle suction and pooled in polypropylene vessels for further examination. analysis of bal fluid bal fluid was transported to the laboratory within h for immediate processing. for cellular analysis, bal fluid was filtered through gauze and adjusted to a concentration of /ml. cells were used for each of cytospin slides. slides were dried by air, fixation was achieved by % acetone. slides were stained by the may-grünwald-giemsa procedure or processed for immunocytochemical analysis. cells were counted for differential cell analysis using light microscopy. the number of macrophages, lymphocytes, neutrophils and eosinophils was expressed as a percentage of the total cell number. episodes of acute deterioration in lung function (mainly decrease in fev , mef ), decrease in po , or newly developed infiltrates in the chest x-ray, were considered as rejection if no pathogen was detected in the bal, and if treatment with corticosteroids proved effective. transbronchial biopsies were not taken routinely. bal was examined routinely for the presence of bacteria, virus or fungus. methods included gram-staining and conventional culture techniques as well as special culture, immunocytochemistry, serologic testing, or pcr. in this way, detection procedures were carried out for general bacteria, mycobacteria, legionella, chlamydia, mycoplasma, pneumocystis carinii, candida, aspergillus, cryptococcus and viruses (paramyxovirus, parainfuenza, hsv, ebv, rsv, adenovirus and cmv). cytospin slides for immunocytochemistry were available from patients. the slides were incubated with a rabbit anti-human il- abbreviations: copd chronic constructive pulmonary disease, a atd alpha antitrypsin deficiency, pph primary pulmonary hypertension, cf cystic fibrosis, ipf idiopathic pulmonary fibrosis, lam lymphangiomyomatosis, ards adult respiratory distress syndrome antibody (product no ± , genzyme, cambridge) at a dilution of : for min at room temperature. after washing, a second antibody (mouse anti-rabbit igg, product no m , dako; hamburg) and a third antibody (anti-mouse igg, product no z , dako; hamburg) were applied, each at a dilution of : for min. finally alkaline phosphatase anti alkaline phosphatase staining procedure was performed (dako). identification of cell type was achieved by microscopical inspection. stained cells were counted and expressed as a percentage of the total count of each cell type. interleukin- was detected using an enzyme immunoassay with no crossreactivity to il- , il- or gm-csf (product no. , immunotech, hamburg). assays were performed as indicated by the instruction manual. briefly, an anti-il- monoclonal antibody-coated microtiter plate was incubated with ml of sample. in a second immunological step, ml of biotinylated monoclonal antibody and a streptavidin-horseradish peroxidase conjugate were added. using tmb as substrate, absorbance was taken at nm. il- concentrations were calculated from a standard curve obtained in the same assay procedure as the sample. the sensitivity of this assay was pg/ml sample. four out of patients who were examined after lung transplantation demonstrated episodes of eosinophilic alveolitis. bal samples were obtained from these patients at different time points. there were bal samples with increase of eosinophils ranging from % to more than % of the total cell number. bal samples demonstrated eosinophilia of % or more. an increase in the number of eosinophils was accompanied by a raised number of neutrophils (fig. ). all patients fulfilled our criteria for indicating acute rejection. in most cases, treatment with steroids during episodes of eosinophilic alveolitis resulted in normalization of the clinical status, significant improvement of lung function, or reduction of infiltrates on chest x-ray. if control bal samples were obtained after treatment, the number of eosinophils was found to be normal or significantly reduced. the course of patient a (transplantation for cystic fibrosis of the lung) is shown in fig. . two of his bal samples showing eosinophilic alveolitis (on ± , not included in the fig., and on ± ) were not followed by treatment. lavages obtained resp. days later demonstrated no significant change in differential cell count. at these points of time, steroid treatments were initiated, leading to improvement in lung function and blood gas analysis. he suffered from recurrent reactivation of cytomegalovirus (cmv) infections after treatment with steroids, showing an increase in cmv ie-antigen positive lymphocytes in peripheral blood samples. cmv reactivation was not associated with elevated counts of eosinophils in bal, compared to times of dormant cmv status. with this patient, episodes of eosinophilic alveolitis ( > % on ± and % on ± , resp.) were accompanied by blood eosinophilia ( %). none of the other episodes of eosinophilia in bal (including all patients) showed blood eosinophilia of more than %. clinical and laboratory data of patient b (transplantation for pulmonary hypertension) are presented in fig. . steroid therapy was initiated (on ± ), because signs of rejection appeared. at that time, the count of eosinophils was %. despite treatment however, the percentage of eosinophils increased to %. complete resolution was achieved by a further application of high dose steroids for days followed by a taper. with this patient, another episode of slightly eosinophilic alveolitis occurred half a year later. at that time, transbronchial biopsies were obtained that showed signs of rejection. in patient c, (transplantation for emphysema) one episode of eosinophilic alveolitis occured that was treated with steroids. this treatment resulted in a reduced but nevertheless elevated count of eosinophils. additionally, high-dose methylprednisolone treatment followed by a taper was administered for days, resultingin normalization of bal differential cell count. in patient d, (transplantation for postinfectious respiratory distress syndrome) very high counts of eosinophils in bal ( > %) were detected during a period of clinical deterioration. treatment with steroids improved her status significantly, however, close followup lavage results were not available. twenty-one of the bal samples of patients with intermittent eosinophilic alveolitis were analyzed for interleukin- (il- ) expression. in all samples il- was detected immunocytochemically on macrophages ( %± % of macrophages). expression on lymphocytes was found in bal samples and ranged between %± % of total lymphocyte count. there was no correlation between the number of lymphocytes expressing il- and the number of eosinophils in bal. in bal samples polymorphnuclear granulocytes (pmn) expressed il- ( %± %). pmn expressing il- were detected only if total number of pmn was increased. elevation of eosinophils was not correlated to the number of pmn expressing il- . there was no immunocytochemical detection of il- in eosinophils. concentration of interleukin- (il- ) was assayed in bal supernatant using elisa technique. il- in native bal fluid was determined at levels between less than pg/ml and , pg/ml. in bal samples without eosinophilic alveolitis the median il- content in unconcentrated fluid was . pg/ml. lavages with an increased number of eosinophils showed il- levels slightly higher at a median of . pg/ml (fig. ) . the difference was statistically not significant (u-test of wilcoxon, mann, whitney). eosinophilic granulocytes are involved in the course of many diseases, mainly allergic, infectious, hematological, and in collagen disorders. pulmonary diseases characterized by increased numbers of eosinophils in bal and tissue are asthma, idiopathic pulmonary fibrosis, acute and chronic eosinophilic pneumonia, churg-strauss syndrome, filarial and fungal infections, and drug reactions [ , , , ] . eosinophils participate in the immunomodulatory cell network. they are capable of both responding to and producing cytokines. they were identified as potent proinflammatory cells, producing reactive oxygen species and releasing toxic granule proteins, such as eosinophil cationic protein and major basic protein [ ] . in the lung, eosinophils are able to degrade connective tissues and cause severe epithelial and microvascular injury. reduced ciliary motility and respiratory epithelial necrosis, as described for asthmatic patients, enhance the risk of infections [ , ] . tissue eosinophilia is involved in allograft rejection after renal, hepatic, pancreatic and cardiac transplanta- [ ] . in renal-and hepatic allografts, episodes of rejection were observed that were associated with peripheral blood eosinophilia [ , ] . our results indicate that eosinophils may play a significant role in lung allograft rejection, too. these findings are in keeping with prior animal and human studies. in a rat-lung allograft transplantation model, bal eosinophilia was found to be a marker of rejection [ ] . histological examination of transbronchial biopsies of patients suffering from rejection showed eosinophils in less than half of the cases, but the number of cells was reduced after treatment with steroids [ ] . in transbronchial biopsies of patients presenting with acute cellular rejection early after lung transplantation, % of the samples displayed eosinophilic infiltrates if rejection was classified as mild. in cases of severe rejection, all samples had eosinophils indicating a correlation between the extent of tissue damage and the number of eosinophils. all cases with intense infiltrates of eosinophils (more than % of infiltrating cells) observed in transbronchial biopsies within one month after transplantation, were associated with rejections. however, there was only one case described exhibiting bal eosinophilia [ ] . riise et al. reported data demonstrating activation of eosinophils during rejection assessed by eosinophil cationic protein in bal, but there was only one case of bal eosinophilia of more than % [ ] . results of our study indicate that after lung transplantation some patients show severe eosinophilic alveolitis during episodes regarded as rejection. to our knowledge, this is the first report about recurrent bal eosinophilia in lung transplant recipients. to validate our findings regarding eosinophils in bal and the diagnosis of acute rejection, a number of prerequisites have to be fulfilled to exclude other mechanisms. firstly, it should be a temporary finding. secondly, simultaneous clinical signs of rejection should occur. thirdly, active infective processes should be excluded. fourthly, response to anti-rejection therapy must be proven [ ] . all four criteria were fulfilled by our patients. it remains uncertain, however, why some patients demonstrate eosinophils in the bal during episodes regarded as rejection after transplantation, and others do not. most of our patients ( out of ) did not produce significant eosinophilia, although most of them showed episodes of rejection by the criteria mentioned above. there may be an association between the extent of rejection and the total number of eosinophils infiltrating affected tissue. differences in adhesion of the eosinophils to the lung tissue may be involved as well. blood eosinophilia was uncommon in our patients, with signs of rejection after lung transplantation suggesting the origin of the process to be the affected lung. peripheral blood eosinophils may be normal during times of rejection, although a relative increase in eosinophil counts is considered a specific marker of rejection [ ] . in our patients, eosinophilic alveolitis was accompanied by an increased number of neutrophils. usually, in acute rejection lymphocytosis is found in up to % of the cases. however, neutrophilic alveolitis is described in patients with acute rejection associated with pulmonary infiltrates on the chest x-ray [ ] . increased numbers of eosinophils may be found in patients with asthma. none of our patients and none of the donors was known to have asthma. eosinophilic alveolitis has been described in angioinvasive aspergillosis after lung transplantation. in one of these cases there were features of acute eosinophilic pneumonia with no proof of aspergillus infection in the first biopsies, and aspergillus was found later [ ] . in patients with asthmatic symptoms, increased ige and eosinophilia in combination with recovery of aspergillus, allergic bronchopulmonary aspergillosis (abpa) must be suspected. however, none of the patients with bal eosinophilia had signs of abpa. increased numbers of eosinophils after lung transplantation have been found in association with coxsackie a and pseudomonas aeruginosa infection as well [ ] . two of our patients had pseudomonas aeruginosa detectable in sputum or tracheal secretion. this pathogen was found consistently, with no association to periods of eosinophilia in bal. treatment with steroids would have been detrimental if pseudomonas were the pathogen causing decrease in clinical parameters of lung function. in one of the patients, corynebacterium jeikeium was detected during an episode of eosinophilic alveolitis, which however persisted after treatment with steroids, and resolution of eosinophilia and symptoms. thus, we think the pathogen was not responsible for the eosinophilia. since ciprofloxin may be related to blood eosinophilia, this antibiotic needs to be considered responsible for bal eosinophilia as well. during one period of eosinophilia in bal, of the patients received ciprofloxin at the same time. however, eosinophilia was restricted to the lung with no blood eosinophilia, which would be unusual for allergic drug reactions. differential diagnosis of eosinophilic alveolitis must include acute eosinophilic pneumonia. criteria defining this disease are essentially the same as the criteria we used for the diagnosis of acute rejection with the exception of bal eosinophilia. however, recurrent episodes of bal eosinophilia ± as we observed in of our patients ± are uncommon in acute eosinophilic pneumonia [ ] . previous reports indicate tissue eosinophilia and activation of eosinophilic markers during episodes of rejection [ , , , , , ] . summarizing our findings regarding acute eosinophilic pneumonia and eosinophilia during rejection, we suspect the eosinophilic alveolitis in lung transplant recipients to be a distinctive form of acute eosinophilic pneumonia indicative for rejection. in one of our cases of eosinophilic alveolitis, transbronchial biopsies were taken, showing signs of acute and chronic rejection, supporting our assumption. however, since this practice was not a common approach when evaluating patients after lung transplantation, we cannot completely exclude different causes in the other cases of eosinophilia in bal. the cytokine il- is known to be involved in the process of eosinophilic inflammation. il- is able to promote differentiation, recruitment and activation of eosinophils [ ] . therefore, in patients who presented eosinophilic alveolitis recurrently, we analyzed bal cells immunocytochemically for il- presence and bal fluid for il- content by elisa. the dominant cells in bal staining positive for il- were macrophages. in addition, il- was detected in neutrophils and t-lymphocytes, but there was no correlation to the number of eosinophils. no il- was detected in eosinophilic granulocytes. studies analyzing il- expression in patients after lung transplantation demonstrated il- mrna in %± % of the samples with a slight increase during episodes of rejection. cell type specification and quantity of il- expression were not examined [ ] . in asthmatic patients, raised expression of il- mrna was detected in bal and in bronchial mucosal cells, where t lymphocytes, especially the cd + th subset, represented a major source of cytokine expression [ , ] . the number of bal cells expressing il- mrna was higher in asthmatic patients who were not treated with steroids, compared to patients who were [ ] . il- appeared to be locally produced, since systemic cytokine levels measured in the blood were low. in patients with asthma, local allergen challenge was performed inducing significant airway eosinophilia [ ] . it was demonstrated that this eosinophilic inflammation was associated with local expression of interleukin in eosinophils indicating an autocrine stimulation. however, while il- seems to be involved in asthma, hypereosinophilic syndrome, eosinophilic cystitis and in eosinophilic heart disease, some studies suggest, that il- expression is not observed in all disorders associated with eosinophilic infiltration, such as crohn's disease [ ] . this might be true as well for eosinophilic inflammation during rejection episodes, since none of our patients demonstrated significant il- levels in unconcentrated bal. even during episodes of significant eosino-philia, il- was detectable at a concentration of only pg/ml bal fluid or lower, although there was a slight difference between episodes of eosinophilia and episodes without eosinophilia (median of . vs. . pg/ ml). the small number of samples analyzed limits the findings with regards to il- concentrations. the difference may become significant in a larger study population. in chronic eosinophilic pneumonitis, increased levels of il- in bal fluid were observed in affected lung segments, corresponding to the extent of pulmonary eosinophilia [ ] . similar results were obtained in vivo, where anti-il- antibody inhibits infiltration of eosinophils in a mouse model [ ] , and in asthmatic patients. in symptomatic asthmatic patients with numbers of eosinophils higher than /ml, il- concentration in bal was elevated to pg/ml, whereas in patients with asymptomatic asthma, or with eosinophilic cell counts lower than , il- was below pg/ml [ ] . il- in the bal fluid of our patients was low even in the presence of high numbers of eosinophils. we therefore hypothesize, that eosinophilic inflammation during rejection episodes may be induced by cytokines different from il- , but known to be involved in eosinophilic inflammation, such as il- , gm-csf and il- [ , ] . in-vitro studies demonstrated saline induced migration of eosinophils into peritoneum mediated by ltb that was released by resident mast cells and macrophages [ ] . this process appears to be mediated either by il- inducing a specific eosinophilic migration, or by il- inducing a mixed migration of eosinophils and neutrophils [ ] . bal differential cell count from our study population showed significant amounts of neutrophils, suggesting the possibility of an il- mediated process. in summary, our observations indicate that after lung transplantation some patients may develop eosinophilic alveolitis similar to acute eosinophilic pneumonia, which may be considered as an acute rejection episode if pathogens causing eosinophilia are excluded. this process does not seem to be mediated by il- . we suggest further studies including transbronchial biopsies for the grading of rejection and exclusion of differential diagnoses to confirm our observation. diagnostic significance of increased bronchoalveolar lavage fluid eosinophils blood and graft eosinophilia as a rejection index in kidney transplant eosinophilic inflammation in asthma eosinophils express interleukin and granulocyte macrophage-colonystimulating factor mrna at sites of allergic inflammation in asthmatics immunohistochemical detection of gm-csf, il- and il- in a murine model of allergic bronchopulmonary aspergillosis allergic granulomatosis, allergic angiitis and periarteriitis nodosa the histological changes in transbronchial biopsy after treatment of acute lung rejection in heart-lung transplants bronchoalveolar lavage and transbronchial lung biopsy during acute rejection and infection in heart-lung transplant patients interleukin synthesis by eosinophils: association with granules and immunoglobulin-dependent secretion bronchoalveolar lavage in allergic asthmatics study of eosinophilia and hepatic dysfunctionas a predictor of rejection in human liver transplantation non-invasive parameters for detection of cardiac allograft rejection cytokine production at the site of disease in chronic eosinophilic pneumonitis immunocytologic analysis of cells obtained from bronchoalveolar lavage in a model of rat lung allograft rejection partcipation of interleukin- and interleukin- in the eosinophil migration induced by a large volume of saline prognostic role of eosinophils in pulmonary fibrosis activation of eosinophils and fibroblasts assessed by eosinophil cationic crotein and hyaluronan in bal. association with acute rejection in lung transplant recipients predominant t h -like bronchoalveolar t-lymphocyte population in atopic asthma prednisone treatment in asthma is associated with modulation of bronchoalveolar lavage cell interleukin- , interleukin- and interferon-g cytokine gene expression recent progress in lung transplantation the role of transbronchial biopsies in the management of lung transplant recipents eosinophilic inflammation is associated with elevation of interleukin- in the airways of patients with spontaneus symptomatic asthma acute eosinophilic pneumonia: histopathologic findings in nine patients secretion of the eosinophil-active cytokines interleukin- , granulocyte/macrophage colony-stimulating factor and interleukin- by bronchoalveolar lavage cd + and cd + t cell lines in atopics asthmatics, and atopic and nonatopic controls association between blood eosinophil counts and acute cardiac and pulmonary allograft rejection epithelial injury by human eosinophils eosinophil activity in bronchial asthma recombinant human interleukin- is a selective activator of human eosinophil function cytokine gene expression in human lung transplant recipients phenotype of cells expressing mrna for th -type (interleukin and interleukin ) and th -type (interleukin and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic and normal control subjects graft eosinophilia in lung transplantation lymphocytic bronchitis/bronchiolitis in lung allograft recipients revision of the working formulation for the classification of pulmonary allograft rejection: lung rejection study group acknowledgement we wish to thank sonja rohweder for her excellent technical assistance. key: cord- -mvgp qfv authors: soccal, p. m.; aubert, j.-d.; bridevaux, p.-o.; garbino, j.; y., thomas; rochat, t.; rochat, t. s.; meylan, p.; tapparel, c.; kaiser, l. title: upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: mvgp qfv background. lung transplant recipients are frequently exposed to respiratory viruses and are particularly at risk for severe complications. the aim of this study was to assess the association among the presence of a respiratory virus detected by molecular assays in bronchoalveolar lavage (bal) fluid, respiratory symptoms, and acute rejection in adult lung transplant recipients. methods. upper (nasopharyngeal swab) and lower (bal) respiratory tract specimens from lung transplant recipients enrolled in a cohort study and undergoing bronchoscopy with bal and transbronchial biopsies were screened using different polymerase chain reaction—based assays. results. bal fluid and biopsy specimens from bronchoscopic procedures performed in patients were analyzed. we also compared paired nasopharyngeal and bal fluid specimens collected in a subgroup of cases. the overall viral positivity rate was . % in the upper respiratory tract specimens and . % in the bal samples (p < . ). we observed a significant association between the presence of respiratory symptoms and positive viral detection in the lower respiratory tract (p = . ). conversely, acute rejection was not associated with the presence of viral infection (odds ratio, . ; % confidence interval, . – . ). the recovery of lung function was significantly slower when acute rejection and viral infection were both present. conclusions. a temporal relationship exists between acute respiratory symptoms and positive viral nucleic acid detection in bal fluid from lung transplant recipients. we provide evidence suggesting that respiratory viruses are not associated with acute graft rejection during the acute phase of infection. flex, and abnormal lymphatic drainage. nevertheless, our knowledge of the clinical effect of respiratory viruses is incomplete, particularly when detected by molecular assays applied to lower respiratory tract specimens. this is in part due to the retrospective design of most available studies, the use of different sampling sites (upper vs lower respiratory tract specimens), and the heterogeneity of diagnostic procedures (conventional vs molecular techniques) [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the direct consequences of any viral infection, subsequent cellular injury and altered host immunity could also initiate a cascade of immunologic events [ ] [ ] [ ] , leading to acute and chronic allograft rejection. although several studies support the association between respiratory viruses and chronic lung rejection [ , , , , , [ ] [ ] [ ] , the link between respiratory viruses and acute rejection has been studied mainly in small case series or by subgroup analysis [ , , - , , ] . on the basis of available evidence, the relationship between viral infection and acute rejection has not been established. the present investigation was specifically designed to assess the epidemiology of respiratory viruses in bronchoalveolar lavage (bal) fluid from lung transplant recipients and to analyze the relationship between these viruses and the presence of acute graft rejection. the most sensitive molecular assay methods were used to identify as many as common respiratory viruses in not only the lower but also the upper respiratory tract. symptoms were carefully and prospectively described, and their association with either respiratory viruses or acute rejection was analyzed. during a -month study period from november through march (covering winter seasons), lung transplant recipients were enrolled in a prospective cohort study [ ] . patients were followed up at the sites of a single transplantation network, including the university hospitals of geneva (geneva, switzerland) and the university hospital of lausanne (lausanne, switzerland). informed consent was required from each participant, and the study was approved by both institutional ethics committees. any lung transplant recipient who underwent bronchoscopy with bal and transbronchial biopsies was eligible, irrespective of the reasons leading to the procedure. on the basis of standardized guidelines, we perform routinely scheduled bronchoscopies at , , , and months after transplantation and yearly thereafter. other indications for bronchoscopy with transbronchial biopsies include unexplained respiratory symptoms, functional deterioration with a у % decrease in the forced expiratory volume in s (fev ), new chest radiologic infiltrate, and control procedure month after treatment of any rejection of grade a or higher. thus, patients could undergo bronchoscopy for a variety of reasons with additional procedures during the study period. for technical reasons (eg, pathologist availability), transbronchial biopsies are not performed during weekends and holidays at our centers. for safety and ethical reasons, no additional bronchoscopy was performed only for the purpose of the study. the bal procedure and the real-time taqman reverse-transcription polymerase chain reaction (rt-pcr) assays for the detection of rna respiratory viruses (influenza viruses a, b, and c; respiratory syncytial viruses a and b; parainfluenza viruses , , , and ; human rhinovirus; enterovirus; human metapneumovirus; and coronaviruses oc , e, nl , and hku ) were performed as described elsewhere [ ] [ ] [ ] . the recently identified bocavirus was added. transbronchial biopsies were performed under fluoroscopic guidance using standard procedures [ ] and analyzed according to published guidelines [ ] by senior pathologists masked to the viral results. pooled nasopharyngeal and oropharyngeal swab specimens were obtained from patients who agreed to the procedure. for technical reasons and reasons of cost ( , rt-pcr assays were performed), the procedure (nasopharyngeal and oropharyngeal swabbing) was limited to % of the cases selected during the entire study period. swabs were immediately placed on appropriate transport medium and stored at Ϫ Њc until further rt-pcr analysis. shortly before each bronchoscopic procedure, a specific case report form was completed and symptoms, reasons leading to the bal procedure, and the presumed diagnosis based on the available clinical data at the time were recorded. rhinopharyngitis was defined as the presence of acute respiratory symptoms with at least acute rhinorrhea with or without additional signs suggesting acute sinusitis and/or acute pharyngitis (sore throat confirmed by the presence of inflammatory signs on clinical examination). flulike illness was defined as the presence of a temperature . Њc plus of the following symptoms: cough, myalgia, sore throat, or headache. when available, lung functions measured - weeks before, at the time of, and - weeks after the bal procedure were recorded. fev and maximal midexpiratory flow of %- % were also recorded. impaired lung function was defined as a у % decrease from the previous value. statistical analysis. each episode was classified into categories according to the respective absence (a or a grade) or presence (a , a , or a grade) of acute rejection and/or respiratory virus. we considered only acute rejection grade a or higher in the analysis, because most centers would not recommend high-dose immunosuppressive treatment for a lower acute rejection grade. the fisher exact test was used to compare the frequency of respiratory symptoms in each category. generalized linear latent and mixed models (stata software, version ; statacorp) were used for analysis of lung functions at each episode to take into account the repeated measures in each patient at the study sites. odds ratios (ors) for acute rejection associated with respiratory viruses were calculated using the same statistical approach for repeated measures. to investigate a potential time-specific relationship between respiratory viruses and acute rejection, we repeated our analyses of episodes restricted to key periods: - months, - months, - months, and months after lung transplantation. seventy-seven lung transplant recipients underwent bronchoscopies. patient characteristics are given in table . all patients received induction immunosuppression with anti-interleukin receptor antibodies (basiliximab) postoperatively, followed by a triple immunosuppression regimen of calcineurin or target of rapa- of the bronchoscopies, ( . %) were performed as routinely scheduled procedures, ( . %) were performed for a new clinical condition, and ( . %) were performed as a control biopsy after treatment for an acute rejection (grade a or higher) episode. some patients included for a routinely scheduled bronchoscopy presented with a new respiratory symptom with or without functional impairment and/or new radiologic infiltrate. taken together, ( . %) of cases had at least new respiratory symptom, and ( . %) had a new radiologic infiltrate. on the basis of the pretest evaluation, acute lung rejection and infection were suspected by the physician in charge, who was masked to microbiological results, in cases ( . %) and cases ( . %), respectively. for the remaining cases ( . %), the clinician did not suspect any particular diagnosis. rt-pcr assays. the rt-pcr viral assays performed on the bal fluid specimens revealed an overall positivity rate of . % ( ). eight bal specimens were discarded ben p cause they had thawed during an electricity power outage. rhinovirus was the most frequently encountered virus, followed by coronaviruses and parainfluenza viruses ( table ). the viral positivity rate for the pooled nasopharyngeal and oropharyngeal specimens tested ( . % of the total study population) was . % ( ). the agreement between upper and n p lower respiratory tract specimens according to each type of viral genus is shown in figure . although influenza viruses, human metapneumovirus, and bocavirus were mostly (or even exclusively for bocavirus) found in the upper airway samples, other viruses (rhinoviruses and respiratory syncytial viruses) were found equally in the upper and lower respiratory tracts. no virus was exclusively found in the lower airways. association between clinical suspicion and final diagnosis. because bal fluid specimens were collected for a variety of clinical conditions, we were able to analyze the association among symptoms, the diagnosis suspected by the physician in charge, and the subsequent presence of a proven upper and/ or lower respiratory tract viral infection. we found a significant association between positive viral nucleic acid detection in bal fluid and the presence of at least new respiratory symptom ( ), in particular cough ( ) and sputum pro-p p . p p . duction ( ). table gives the clinical findings associated p p . with virus positivity for upper and lower tract specimens. physicians in charge suspected an infection in ( . %) of the cases with virus-positive bal fluid, compared with ( . %) of the cases that were virus negative. a history of rhinopharyngitis was documented in ( . %) of cases with a virus-positive nasopharyngeal swab specimen, compared with ( . %) of of those that were negative. of biopsy specimens, ( . %) showed no evidence of acute rejection, ( . %) revealed minimal (a ) rejection, and ( . %) were graded a or higher. similar to the analysis given in table , we first tested the accuracy of the pretest clinical evaluation performed by the physician in charge. when acute rejection was suspected by the clinician in charge, this was histologically confirmed in . % of cases in the absence of respiratory virus and in . % of cases when a respiratory virus was simultaneously identified in bal fluid (table ). consistent with the results shown in table , the presence of any respiratory symptoms, cough, or sputum was significantly associated ( for all) with the presence of a viral p ! . infection in bal fluid, irrespective of the presence or absence of acute rejection. impairment of lung function related to acute rejection was also significantly worsened by the presence of a viral infection (figure ). in patients with simultaneous acute rejection and lower respiratory tract viral infection, the fev recovery rate was significantly slower than in patients who had acute rejection without simultaneous viral infection ( ). p ! . we found that the or for acute rejection was ! in the presence of a viral infection for each of the periods studied (or for any period, . ; % confidence interval [ci], . - . ). the overall rate of viral infection in grade a and a cases was . % ( of ), compared with . % ( of ) in grade a or higher. thus, patients with a lower acute rejection grade were twice as likely to be positive for viral infection than those with a higher rejection grade (or, . ; % ci, . - . ). when repeating these analyses with the presence of virus in the upper respiratory tract as a predictor of acute rejection, we did not find an association between upper respiratory tract viral infection and acute rejection (or, . ; % ci, . - . ). in a supplementary analysis, we verified the occurrence of acute rejection during a -day and -day period after baseline bronchoscopy. at and days, the probability of acute rejection was associated with the presence of acute rejection at baseline using an extensive panel of molecular assays, we were able to show that . % of prospectively collected bal fluid specimens from adult lung transplant recipients were positive for at least respiratory virus. on the basis of a pretest clinical evaluation, patients testing positive were significantly more likely to present with respiratory symptoms ( . %, compared with . % with no identified virus). all analyzed individual symptoms were systematically more frequent in the presence of a respiratory virus, particularly cough and sputum, which were up to times more common. consistent with these observations, the pretest clinical evaluation performed by the physicians in charge considered that an infection was more likely in those with a final positive viral detection. furthermore, lung function proved to recover significantly slower in the presence of a respiratory virus. all these findings corroborate that a positive association exists between positive viral nucleic acid detection in bal fluid and the presence of an acute respiratory illness in lung transplant patients. this is also concordant with other investigations that have linked the detection of respiratory viruses by rt-pcr to symptoms [ , , , , , , ] . however, our investigation differs from these studies by its prospective design, the large panel of respiratory viruses screened, the systematic use of bal specimens, and the integration of pretest clinical evaluations performed by the physicians in charge. many of the previous studies limited the detection strategy to upper respiratory tract specimens and used an array of molecular tools that was often restricted to a subgroup of viruses (eg, rhinoviruses, coronaviruses, or bocaviruses were not systematically tested). finally, the link with pretest clinical conditions was not detailed in many of these reports. because of the availability of lung biopsy specimens in all cases, we were also able to assess the presence of acute rejection according to the presence of viral infection. a relationship between acute viral infection and subsequent acute rejection has been reported in small case series, but this possible association has not been appropriately confirmed. viral infection might trigger a chain of immunologically mediated events, leading to subsequent rejection or lung dysfunction. this is important because several studies have identified viral infection as a distinct risk factor for the development of bronchiolitis obliterans syndrome and chronic graft dysfunction [ , , , , , , , ] . our investigation had the capacity to address this question because we were able to analyze a high number of lung biopsy specimens together with viral screening at the level of the lower respiratory tract. the biopsy analysis revealed that viral respiratory tract infections were not associated with simultaneous acute rejection. furthermore, we could show that the probability of acute rejection did not increase and days after a lower airways respiratory viral infection. when present, however, viral infections caused more severe lung dysfunction and significantly hampered the short-term functional recovery in the case of concomitant acute rejection. because the clinician in charge was masked to any viral result, a positive rt-pcr result did not modify the treatment of simultaneous acute rejection. thus, a less aggressive treatment of acute rejection in the presence of a virus cannot explain the slower recovery of lung function in these patients. this suggests that respiratory viruses per se do not promote acute rejection during the acute phase but could certainly worsen lung function and impair recovery from acute rejection episodes. paradoxically, we even observed a negative association between an episode of respiratory viral infection and the subsequent cumulative risk of developing acute rejection. this should not be considered a protective effect of viral infections; more likely, it is a lack of an association or a chance effect, and we refrain from drawing any other conclusions. however, this strongly suggests that when analyzing biopsy specimens according to reference guidelines [ ] , pathologists should not be misguided by the presence of a viral infection. of note, our study design limited follow-up to a few weeks, and we cannot exclude the possibility that the viral infection triggered a chain of immunologically mediated events, leading to subsequent rejection or lung dysfunction. this is important because several studies have identified viral infection as a distinct risk factor for the development of bronchiolitis obliterans syndrome and chronic graft dysfunction [ , , , , , , , ] . our investigation also provided the unique opportunity to compare viral detection in the upper (pooled nasopharyngeal and oropharyngeal swabs) and lower respiratory tract in a large number of paired specimens. the positivity rate in the upper respiratory tract was . %, compared with . % in the lower tract. this difference in recovery rate is similar to that observed in other smaller studies [ ] , but, to the best of our knowledge, few or none of the previous investigations systematically analyzed paired specimens collected during the same procedure. an important observation is that when recovered only in the upper respiratory tract, respiratory viruses were less likely to be associated with lower respiratory symptoms. another interesting observation is that only . % of negative nasopharyngeal specimens were associated with a discordant positive bal viral screening, thus suggesting a high negative predictive value. however, this value should be balanced with the relatively low prevalence of each individual respiratory virus in bal fluid specimens and the possible technical issues related to nasopharyngeal and pharyngeal swabbing that could negatively affect the recovery rate. our population was first selected on the basis of the need to perform a bal procedure; for this reason, we caution that a lower respiratory tract viral infection in lung transplant recipients cannot be definitely ruled out by a nasopharyngeal swab. yet this could be a reasonable initial screening strategy that needs to be confirmed in further studies and individually for each type of virus. in conclusion, our study demonstrates that there is a temporal relationship in lung transplant recipients between the emergence of acute respiratory symptoms and positive respiratory viral nucleic acid detection in bal fluid specimens. when detected only in the upper respiratory tract, viral infections are less likely to be associated with respiratory symptoms and graft dysfunction. we also provide solid evidence suggesting that respiratory viruses per se do not promote acute graft rejection, at least during the acute phase of infection, but that they do worsen graft function recovery when simultaneously present with acute rejection. clinical impact of communityacquired respiratory viruses on bronchiolitis obliterans after lung transplant impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients respiratory viruses and chronic rejection in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus respiratory viral infections in transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients absence of human bocavirus in bronchoalveolar lavage fluid of lung transplant patients diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin infectious etiology of bronchiolitis obliterans: the respiratory viruses connection: myth or reality? characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance critical role for the chemokine mcp- /ccr in the pathogenesis of bronchiolitis obliterans syndrome community acquired respiratory viral infections after lung transplantation: clinical features and long-term consequences parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function the epidemiology of parainfluenza virus infection in lung transplant recipients treatment of respiratory syncytial virus pneumonia in a lung transplant recipient: case report and review of the literature influenza virus infection in adult solid organ transplant recipients respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection swiss paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study persistent hepatitis c viremia predicts late relapse after sustained response to interferonalpha in chronic hepatitis c lung rejection study group. revision of the working formulation for the classification of pulmonary allograft rejection influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients we thank patricia suter, sandra van belle, jean-marc fellrath, and lara turin for their excellent technical assistance and rosemary sudan for editorial assistance.financial support. swiss national science foundation (grant b- to l.k.).potential conflicts of interest. all authors: no conflicts. key: cord- -a tdgns authors: abul, h.; abul, a.; khan, i.; matthew, t.c.; ayed, a.; al-athary, e. title: levels of il- and myeloperoxidase in the lungs of pneumonia patients date: journal: mol cell biochem doi: . /a: sha: doc_id: cord_uid: a tdgns interleukin- (il- ) is considered as the major polymorphonuclear neutrophils (pmns) chemoattractant cytokine in lung diseases such as asthma and adult respiratory distress syndrome (ards). however, controversial results were obtained regarding the involvement of il- in the pathogenesis of pneumonia. this study examines the role of il- in the recruitment and activation of pmns in the lung of pneumonia patients. the interesting aspect of this study is that it is a site- specific analysis of the infected and uninfected lungs of the same patient. the level of il- mrna, protein and myeloperoxidase present in the cells of the bronchioalveolar lavages (bals) taken from the areas of known pneumonic consolidations on chest x-ray (infected lung) are compared with the bals obtained from areas of no obvious infiltrate (non-infected lung). the results obtained from the infected and non-infected lungs of pneumonic patients were further compared with that of a control group of non-smoking patients. the level of il- mrna and protein were determined by rt-pcr and elisa respectively. there was a significant increase in the level of il- mrna in the infected lung as compared to its level in the non-infected lung (p < . ). in correlation with the increase in mrna, il- protein concentrations in bal fluids from the infected lung were fold higher than those taken from the non-infected lung (p < . ). this pattern was also consistent with mpo activity in the bals ( . fold more mpo activity in the infected lung as compared to that of the non-infected lung), indicating that il- is directly implicated in neutrophil accumulation that follows acute respiratory infection. the results of the present study, therefore, indicate the involvement of il- in the pathogenesis of pneumonia. chemokines constitute a large family of regulatory cytokines that play a central role in immunological processes. the accumulation and appearance of polymorphonuclear neutrophils (pmns) in the tissue may be considered as an initial marker of acute inflammatory reaction [ ] . neutrophils participate in the host response to a number of infectious and non-infectious diseases and in leukocyte migration [ ] [ ] [ ] [ ] [ ] . they contain cytoplasmic granules that function in storage of bioactive neuromolecules (specific or secondary granules) or in fusion with phagosomes (azurophilic or primary granules). the azurophilic granules contain a variety of enzymes including myeloperoxidase, muraminidase, cathepsin a, d, e, g, ′-nucleotidase, β-galactosidase, elastase, collagenase, azurocidin and the defensins hnp- , hnp- , and hnp- , arylsulfatase, α-mannosidase, n-acetyl-β-glucosaminidase, β-glucuronidase, acid β-glycerophosphatase and cationic peptides. the specific granules on the other hand contain vitamin-b -binding protein, neutral proteases, lactoferrin, alkaline phosphatase, lysozyme, and probably collagenase [ ] . although the mechanisms that regulate the release of substances from both the granules are almost the same, there are certain specific stimuli such as il- and zymosan that induce the release of substances from secondary granules [ ] . thus il- functions as a potent chemotacting as well as degranulating agent. recently it has been shown that depletion of neutrophils using anti-rat neutrophil antiserum reduced subsequent development of chronic delayed type hypersensitivity reactions [ ] . this study clearly demonstrates the importance of neutrophil derived factors for monocyte and lymphocyte mobilization. furthermore, it is shown that neutrophils produce a number of low molecular weight factors such as leukotriene b (l bt ) that attract more neutrophils and monocytes to the inflammatory site [ ] . t-lymphocytes have also been shown to migrate in response to il- both in vivo and in vitro [ , ] . there is a clear involvement of il- in the pathophysiology of various respiratory diseases [ ] [ ] [ ] [ ] [ ] . in asthma, airway inflammation with eosinophils, lymphocytes and neutrophils is a characteristic feature [ ] [ ] [ ] . in correlation with this cellular migration, there is an increase in the level of il- in the serum, tissue and bal of asthmatics. similar to that in asthma, the involvement of il- has been well investigated in adult respiratory distress syndrome (ards). however, the role of interleukin in the development of pneumonia is controversial [ ] . although, an increase in the level of il- is a good indication of the inflammatory process, this information does not contribute much to clinical diagnosis. to our knowledge, no data is available on the production of il- in bal fluid from the same patient (i.e. infected and non-infected lung). therefore this study is designed to measure the site-specific increase in the level of il- in the lung of patients with bacterial pneumonia as compared to that of the non-smoking control group. the level of il- mrna and protein present in the bal obtained from subsegmental bronchi of experimental and control group of patients were determined by rt-pcr assay and enzyme immunoassay respectively. in this study we also determined the level of myeloperoxidase activity in the cells collected from ml of bal each from the infected and non-infected lung. myeloperoxidase, a secreted heme protein, is an attractive candidate for monitoring phagocyte mediated cellular damage [ , ] . the study was performed on patients with bacterial pneumonia who were admitted to the chest diseases hospital in kuwait. all patients underwent medical and laboratory examinations. the control group consisted of non-smoking patients among which patients were with chronic cough, with hemoptysis and normal chest x-ray and with old fibrotic shadows (table ) . bals were obtained first from the area of known pneumonic consolidations on chest x-ray (infected lung) followed by bals from other areas with no obvious infiltrate (non-infected lung) of the same patient. bal fluids were collected from the pneumonic patient and the control group after admission to the hospital, using sterile techniques and routine respiratory care. the bronchoscope was advanced into a subsegmental bronchus. lavage was performed using ml aliquots of warmed normal saline, introduced by a syringe through bronchoscopic aspiration port. a total volume of - ml saline was infused sequentially and the volume of the lavage fluid retrieval (approximately ml) was pooled and transferred immediately into sterile pre-chilled polypropylene tubes. the pooled fluid was then filtered through one layer of sterile gauze and centrifuged at rpm for min at °c. following centrifugation, ml of supernatant was taken into a sterile polypropylene tube and stored at - °c until assayed. total rna was extracted from cells contained in ml of the bal obtained from the infected and non-infected lung, using the method of chomczynski and sacchi [ ] . briefly, the method is as follows. cells were lysed in . ml of mguanidinium isothiocyanate. the lysates were then acidified by adding µl of m sodium acetate at ph . . subsequently, . ml of water saturated phenol and . ml chloroform were added to the cellular lysate followed by shaking at °c for min. lysates were spun in the cold for min and the supernatants were collected and extracted again with phenol-chloroform. the supernatants were finally extracted with chloroform and the aqueous layer was collected. rna was precipitated with absolute ethanol [ ] . the precipitate was further centrifuged and the pellet was air-dried. the rna pellet was then dissolved in µl diethylpyrocarbonate (depc) treated water. concentration of rna was determined at / nm optical absorbance. aliquots ( µg) of total rna were annealed with ng of oligo dt primer by heating at °c for min followed by its slow cooling to °c. reverse transcription was carried out using units of avian myeloma virus (amv) reverse transcriptase and units of rna guard following the conditions described [ ] . reverse transcription reaction was carried out in µl total volume and an aliquot of - µl from this cdna was amplified for cycles using the following pcr amplification parameters: denaturation °c × sec, annealing °c × sec and extension °c × sec. the mgcl was used at a concentration of . mmol/l. the pcr amplification reaction was carried out in presence of pmol each of upstream ( ′-gga acc att ctc act gtg tg- ′) and down stream ( ′-ctc ttc aaa aac ttc tcc aca a- ′) il- specific primers using unit of amplitaq enzyme in a thermocycler. these primers were synthesized based on human il- cdna sequence information [ ] . pcr products were analyzed on % polyacrylamide gel electrophoretically [ ] , stained with ethidium bromide and photographed with a gel documentation system (stratagene). all experiments were carried out under rnase free conditions and the solutions and glassware were made rnase free with depc treatment and or by autoclaving. heat sensitive solutions were made in depc treated and autoclaved water followed by their filtration through . µ size millipore filters. the concentrations of il- in plasma and bal fluid supernatants were assayed in duplicate, using a quantitative immunometric, 'sandwich' enzyme immunoassay technique with a detection limit of . pg/ml (amersham, uk). level of myeloperoxidase activity was estimated in the cells collected from ml of bal from the infected and non-infected lung. the method was essentially the same as described earlier [ ] . cells were pelleted by centrifugation at °c and were homogenized in ml of hexadecyltrimethylammonium bromide buffer, containing mm hexadecyltrimethylammonium bromide and mm kpo , ph . . samples were homogenized with polytron for min and were kept cold on ice. the lysates were subsequently frozen in liquid nitrogen and thawed once. the lysates were then centrifuged for min in cold at , rpm and the supernatants were used to estimate the level of mpo activity. aliquots of µl supernatant were mixed with µl of odianisidine hcl (sigma) solution containing . mg of odianisidine hcl, ml of distilled water, ml of kpo buffer, ph . , and µl of % h o . absorbance was recorded at nm, every sec for min using beckman du spectrophotometer. the enzyme activity was calculated (units/min/ml) by dividing the rate of the change in the absorbance by the extinction coefficient, . × - . enzyme unit is defined as the conversion of µmol of h o per min per ml of alveolar lavage at room temperature. under these conditions, the residual activity in the pellet was < %. commutations were performed using the statview . statistical package with macintosh centris computer. results are expressed as means ± s.e.m. the differences between groups were analyzed by student's t-test. the differences between the groups were considered significant if p < . . in equal amounts of total cellular rna, there was a significantly higher level of il- mrna in the infected lung as compared to that of the non-infected lung (p < . ; fig. , lane ). before estimating the changes in the level of il- mrna, we characterized the identity of il- pcr fragment ( bp). for this purpose we employed hindiii restriction enzyme. this enzyme cut the il- pcr fragment ( bp) of free and complex il- in the blood as well as the bronchial mucosa [ ] , suggesting that free il- may have a role in the activation of eosinophils. various other convincing studies suggest that il- is an eosinophil and neutrophil chemoattractant [ ] [ ] [ ] [ ] . it has been shown that il- plays a major role in adult respiratory syndrome (ards) [ ] [ ] [ ] . on the other hand, certain studies could not find a correlation between the percentage of pmns and the concentration of il- in bal fluid of patients with ards [ ] , suggesting that in addition to il- there may be other chemoattractant agents that are involved in transendothelial migration of pmns. several investigators have demonstrated that bal fluids obtained from patients with pulmonary infection, contain potent chemotactic factors, such as the complement peptide c a and leukotriene-b (ltb ) [ ] . in guinea pigs, exogenous il- administration has been shown to recruit neutrophils in the airway lumen [ ] . in addition, in vivo and in vitro studies have shown that il- induces the release of t-lymphocyte chemoattractants from neutrophil [ ] . in vivo studies in mice with a targeted deletion of il- receptor homologue has shown that the total number of recruited cells to the airway lumen following a single antigen challenge was significantly low as compared to the wild type [ ] . in consistent with the above studies, in the present study, high concentrations of il- were found in bal fluids taken from the infected lung of patients with bacterial pneumonia. these results are in agreement with other investigators [ , ] , who reported high levels of il- in bal fluids of patients with different lung diseases. since alveolar macrophages are the major source of il- in the lung, the local production of il- by these cells may be responsible for the recruitment of pmns into the pulmonary interstitial or air space in a variety of lung diseases. further studies are required to determine the relationship between the severity of the concentrations of il- in bal fluids taken from patients with bacterial pneumonia were always high as compared to control group (p < . ; fig. ). furthermore, in all patients the levels of il- in bal obtained from the infected lung were fold higher than those from the non-infected lung . ± to . ± pg/ml, n = (p < . ). this pattern of increased expression of il- mrna and protein was consistent with mpo activity in the lavages. in the cells from the equal amount ( ml) of alveolar lavages there was . times more mpo activity ( . units/min/ml) in the right lung as compared to the level ( . units/min/ml) in the left lung (fig. ). recent reports have considered il- as the most potent and major pmn chemoattractant factor in lung diseases [ ] [ ] [ ] [ ] [ ] , including ards and pneumonia [ ] , cystic fibrosis [ ] , human immunodeficiency virus (hiv)-infected patients with pneumocystis carinii pneumonia, bacterial pneumonia, or tuberculosis [ ] . several studies have convincingly shown that il- plays a key role in the pathobiology of asthma [ ] [ ] [ ] . presence of il- has been demonstrated in the bronchioalveolar fluid (bal) of the patients with asthma [ ] [ ] [ ] . furthermore, in asthmatics, there was an increase in the level lung diseases and the alteration of il- in bal fluids. nevertheless, the data presented clearly show that the concentration of il- in bal fluid from pneumonic patients increased in the infected lung. in the lungs, il- appears to be the primary chemoattractant for neutrophils [ ] . in addition to various cell types [ ] , il- is also synthesized and released by neutrophils [ ] . thus, neutrophils contribute to the recruitment of additional neutrophils in an autocrine manner, by the synthesis and release of il- . several studies suggest that il- also functions as a chemoattractant for eosinophils [ ] [ ] [ ] [ ] . it has been shown that major basic protein (mbp), a . kd protein located in the crystalloid core of eosinophil secondary granules, stimulates the production of il- through transcriptional and posttranscriptional events [ , ] . in neutrophils, mbp stimulated il- production occur post-transcriptionally through stabilization of il- mrna [ ] . in a recent study it has been noticed that type specific consequences of lung infection may be due to the type specific differences in the induction of cytokines by various infectious agents [ ] . in contrast to type adenovirus, type adenovirus stimulated the production of il- in human lung alveolar epithelial cell line (a cells) and primary human fetal lung fibroblasts (gm cells). the regulation of il- production, in these cells, occurred at the transcriptional level and at the level of message stability [ ] . while adenovirus type increased endogenous il- specific mrna, both serotypes (type and type ) enhanced stabilization of il- mrna [ ] . the data presented in our study shows that there was a significant increase in the level of il- mrna in the infected lung as compared to its level in the non-infected lung (p < . ). in correlation with the increase in mrna, il- protein concentrations in bal fluids from the infected lung were fold higher than those taken from the non-infected lung (p < . ). the mechanism of il- specific mrna increase (transcriptional or post-transcriptional) in this study need to be elucidated. the techniques that are commonly used to quantitate inflammatory cells during the development of various diseases in the lungs include histological analysis and ex vivo radiolabeling of leucocytes and quantification of their accumulation in the lungs by counting. these techniques, however, are labor and time intensive and have practical limitations [ ] [ ] [ ] [ ] [ ] . to overcome the limitations of histological and radiolabeling studies, in the present study, we have measured the myeline peroxidase (mpo) activity, in order to quantify the neutrophil accumulation. the pattern of mpo activity in the bals ( . fold more mpo activity in the infected lung as compared to that of the non-infected lung) was consistent with the level of il- mrna and protein. the results of the present study, therefore, indicate a site-specific involvement of il- in the pathogenesis of pneumonia. role of neutrophils and mononuclear phagocytes in host defense and inflammation mechanisms of lysosomal enzyme release from human leukocytes: microtubule assembly and membrane fusion induced by a component of complement the ability of chemotactic factors to induce lysosomal enzyme release. i. the characteristics of the enzyme release, importance of surfaces and the relation of enzyme release to chemotactic responsiveness cytochalasin b: effect of lysosomal enzyme release from human leukocytes the structure activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils some interrelations of neutrophil chemotaxis, lysosomal enzyme secretion, and phagocytosis as revealed by synthetic peptides the development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules sequential degranulation of the two types of polymorphonuclear leukocyte granules during phagocytosis of microorganisms modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, rp- . i. inhibition by rp- treatment of the priming and effector phases of delayed type hypersensitivity to sheep red blood cells in rats the biologically active leukotrienes properties of the novel proinflammatory supergene 'intercrine' cytokine family the neutrophil-activating protein (nap- ) is also chemotactic for t lymphocytes inflammatory determinants of asthma severity: mediator and cellular changes in bronchoalveolar lavage fluid of patients with severe asthma interleukin in bronchoalveolar lavage of asthmatic and chronic bronchitis patients production of interleukin- , rantes and mcp- in intrinsic and extrinsic asthmatics il- is a potent eosinophil chemoattractant interleukin- in airway inflammation in patients with asthma and chronic obstructive pulmonary disease predominant th -like bronchoalveolar t-lymphocyte population in atopic asthma eosinophil recruitment is associated with il- , but not with rantes, twenty-four hours after allergen challenge eosinophilic inflammation in asthma high levels of interleukin- in the blood and alveolar spaces of patients with pneumonia and adult respiratory distress syndrome oxygen metabolism and toxic properties of phagocytes leukocytic oxygen activation and microbicidal oxidative toxins single-step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction molecular cloning. cold spring harbor laboratory altered expression of sodium pump isoforms in the inflamed intestine of trichinella spiralis-infected rats genomic structure of the human monocyte-derived neutrophil chemotactic factor il- polymerase chain reaction assay of mrna using s rrna as internal standard measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker interleukin- concentrations are eluted in bronchoalveolar lavage, sputum and sera of children with cystic fibrosis interleukin- and granulocyte colony-stimulating factor in bronchoalveolar lavage fluid and plasma of human immunodeficiency virus infected patients with pneumocystis carinii pneumonia, bacterial pneumonia, or tuberculosis free and complexed interleukin- in blood and bronchial mucosa in asthma cj: interleukin- -induced human peripheral blood b-lymphocyte chemotaxis in vitro il- is a potent eosinophil chemoattractant t -lymphocyte recruitment by interleukin- . il- -induced degranulation of neutrophils releases potent chemoattractants for human t lymphocytes both in vitro and in vivo il- -induced tlymphocyte migration: direct as well as indirect mechanisms elevated levels of nap- /interleukin- are present in the airspace of patients with adult respiratory distress syndrome and are associated with increased mortality pivotal role of interleukin- in the acute respiratory distress syndrome and cerebral reperfusion injury inhibition of neutrophil-mediated acute lung inflammation injury by an antibody against interleukin- (il- ) neutrophil chemotactic factors in bacterial pneumonia sensory neuropeptides are not directly involved in bronchial hyperresponsiveness induced by interleukin- in guinea-pigs in vivo interleukin- receptor modulates ige production and b-cell expansion and trafficking in allergen-induced pulmonary inflammation interleukin- (il- ): the major neutrophil chemotactic factor in the lung chemokines, inflammation and the immune system cytokine-induced neutrophil-derived interleukin- biology of eosinophils allergy-principles and practice post-transcriptional regulation of gro, and il- mrnas by il- type-specific induction of interleukin- by adenovirus il- is a potent inducer of eosinophil accumulation in rat skin: inhibition of response by a platelet-activating factor antagonist and an anti-human il- antibody the accumulation of in-eosinophils induced by inflammatory mediators, in vivo increased expression of cd b and functional changes in eosinophils after migration across endothelial cell monolayers induction of low density and up-regulation of cd b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils key: cord- -mdyu ca authors: pesci, alberto; majori, maria title: il lavaggio broncoalveolare nelle pneumopatie infiltrative diffuse date: journal: pneumologia interventistica doi: . / - - - - _ sha: doc_id: cord_uid: mdyu ca le pneumopatie infiltrative diffuse costituiscono un gruppo eterogeneo di malattie caratterizzate, istologicamente, dalla presenza di un danno a carico della parete alveolare che puÒ essere infiltrata da cellule infiammatorie/neoplastiche/fluidi/tessuto connettivo. si parla di forme “diffuse” per sottolineare l’interessamento non solo dell’interstizio, ma anche delle strutture acinari e bronchiolari. indagini immunocitochimiche possono essere utilizzate per la individuazione di cellule cd a (sospetto di granulomatosi polmonare a cellule di langerhans). in citofluorimetria il panel di anticorpi monoclonali utilizzati routinariamente sono il cd (linfociti t), cd /cd (linfociti b), cd (linfociti t helper), cd (linfociti t suppressor), cd cd (natural killer). solo in casi particolari (quando i linfociti b sono superiori all' %) si quantificano le catene leggere di superficie kappa e lambda. in soggetti sani non fumatori la cellularità del bal è di circa - cellule/ml e la popolazione cellulare predominante sono i macrofagi alveolari che costituiscono l' %- % delle cellule totali. i linfociti costituiscono il %- %, circa il % sono linfociti t con un rapporto cd /cd dell' , - , l' % sono linfociti b e il %- % sono cellule natural killer. i polimorfonucleati neutrofili (pmn) sono l' %- %, gli eosinofili sono < % così pure i mastociti [ ] . in individui fumatori si osserva aumento della cellularità totale ( - cellule/ml) e modesto incremento della popolazione neutrofila ( %- %) [ ] . in pazienti con pneumopatie infiltrative diffuse possono verificarsi variazioni nella resa e nella conta cellulare differenziale; peraltro, anche variabili quali l'età, abitudine tabagica, assunzione di farmaci, tecnica di esecuzione del bal, tecnica di trattamento del bal possono influenzarne il reperto. la fibrobroncoscopia può provocare traumatismi con conseguente presenza di abbondanti emazie. un trattamento del campione o una colorazione non adeguati possono condizionare l'identificazione cellulare al microscopio. campioni non soddisfacenti contengono meno di due milioni di cellule in totale, cellule epiteliali ciliate > %, cellule epiteliali piatte oro-faringee > %, essudato mucopurulento, eccessive emazie da traumatismo, cellule degenerate per cattiva conservazione. la determinazione di componenti non cellulari (immunoglobuline, mediatori della flogosi, citochine etc.) è complessa e l'utilizzo controverso a causa della variabilità del recupero. queste componenti vengono espresse come unità/mg di albumina o come unità/ml di bal. il bal nelle pneumopatie infiltrative diffuse è utilizzato a fini clinici nella diagnostica, staging, monitoraggio e terapia (proteinosi alveolare). il bal è diagnostico in caso di malattie infettive, malattie neoplastiche, proteinosi alveolare e granulomatosi a cellule di langerhans polmonare. in questo capitolo faremo solo pochi cenni all'utilizzo del bal nelle forme infettive in quanto già esaminate in un altro capitolo. l'isolamento dei seguenti germi nel bal risulta essere diagnostico: pneumocystis jiroveci (fig. la valutazione citologica delle cellule ottenute con il bal può essere utile nella diagnosi di tumori polmonari periferici (non visibili endoscopicamente) sia primitivi che secondari, infatti la sua resa diagnostica è del %- % [ ] . nel carcinoma bronchiolo-alveolare l'analisi del sedimento del bal consente spesso di ritrovare cellule neoplastiche alveolari ben differenziate (fig. ) ; tale reperto però non consente di differenziarlo dall'adenocarcinoma primitivo o dall'adenocarcinoma metastatico polmonare. la resa diagnostica varia, a seconda delle casistiche dal % al % [ , ] . nella linfangite carcinomatosa il sedimento del bal permette spesso di osservare la presenza di cellule neoplastiche ( %- %) ed un aumento aspecifico della popolazione linfocitaria [ , ] . va segnalato che i pneumociti reattivi ed iperplastici di ii tipo che si osservano nel sedimento del bal in corso di diverse polmoniti interstiziali idiopatiche e nella fase organizzativa del danno alveolare diffuso possono mostrare atipie tali da essere confusi con cellule tumorali (fig. ) . . sedimento di bal in soggetto immunodepresso con polmonite da pneumocystis jiroveci. accanto ad un macrofago alveolare è presente essudato con aspetto schiumoso. l'aspetto schiumoso è determinato dalla presenza di cisti sia otticamente vuote sia contenenti trofozoiti (may grunwald giemsa,x ) sedimento di bal in soggetto affetto da carcinoma bronchioloalveolare. immersi in un tappeto di macrofagi alveolari frammisti a pochi linfociti,si osservano due aggregati di cellule ipercromatiche con aspetti di atipia (papanicolau,x ) il lavaggio broncoalveolare nelle pneumopatie infiltrative diffuse occasionalmente il bal può essere utile nella diagnosi di linfoma polmonare rivelando la presenza di un aumentato numero di linfociti che opportunamente studiati rivelano aspetti neoplastici [ , ] . nella proteinosi alveolare il bal può evitare la necessità di una biopsia in quasi tutti i casi. va considerata la diagnosi di proteinosi alveolare se, nell'opportuno contesto clinico-radiologico, già all'analisi macroscopica, il bal si presenta opaco e lattescente [ ] . la microscopia ottica evidenzia un sedimento cellulare caratterizzato da poche cellule, grandi corpi acellulati su uno sfondo di materiale amorfo eosinofilico. il materiale proteinaceo è tipicamente positivo alla colorazione pas e negativo a quella con alcian blue. i macrofagi presenti sono ingolfati da materiale pas-positivo (fig. ) . infine, l'esame in microscopia elettronica rivela la presenza di strutture lamellari concentriche (corpi lamellari). tutte queste caratteristiche risultano essere diagnostiche [ , ] . il bal è caratterizzato da un aumento della cellularità totale, della percentuale dei neutrofili e, talora, degli eosinofili, tale reperti sono però aspecifici. risulta diagnostico, invece, nel giusto contesto clinico-radiologico, il riscontro di una percentuale di cellule di langerhans (cd +) superiore al % (fig. ) [ , ] .va comunque segnalato che le cellule cd + possono essere ritrovate in numero aumentato nel bal di soggetti forti fumatori [ ] o con altre pneumopatie infiltrative diffuse [ ] , in tali condizioni tuttavia esse difficilmente superano il %. al contrario un numero di cellule cd + normale non esclude la diagnosi di granulomatosi polmonare a cellule di langerhans (sensibilità di circa il %) [ ] . . sedimento di bal in soggetto affetto da granulomatosi polmonare a cellule di langerhans.accanto ad alcuni macrofagi debolmente colorati si apprezzano due cellule mononucleate (con ampia piega nucleare) con intensa colorazione immunocitochimica rossa per l'anticorpo monoclonale anti-cd .tale reperto risulta diagnostico quando il numero delle cellule cd + supera il % (colorazione immunocitochimica con anticorpo monoclonale anti-cd ,rivelatore fast red) in altre condizioni, quali la sarcoidosi, polmonite da ipersensibilità, pneumoconiosi, alveolite emorragica, polmonite eosinofila, danno polmonare da farmaci, connettiviti, danno alveolare diffuso (dad), bronchiolite obliterante-polmonite organizzativa e polmoniti interstiziali idiopatiche (nsip, uip), il bal non fornisce un reperto patognomonico, ma, nel giusto contesto clinico-radiologico, può essere di ausilio diagnostico. addirittura in talune occasioni il bal può ridirigere la diagnosi verso patologie che non erano ancora state prese in considerazione nella diagnostica differenziale. il sedimento del bal è caratterizzato da un aumento della cellularità totale e della percentuale dei linfociti (> %) (tabella ), anche se il reperto di linfocitosi non è né sensibile né specifico. i linfociti sono di fenotipo prevalente cd + per cui si riscontra un rapporto cd /cd aumentato. un rapporto cd /cd > , ha una sensibilità del % ed una specificità del %, ed è di ausilio diagnostico nei casi in cui non è possibile la conferma istologica [ , ] (tabella ). una ratio cd /cd < ha un valore predittivo negativo del %. tuttavia è segnalato che circa il % dei pazienti con sarcoidosi possono avere nel bal una ratio cd /cd normale o ridotta [ ] . la diagnosi risulta essere improbabile anche quando nel sedimento del bal si osservano neutrofili > % ed eosinofili > %. i classici reperti di linfocitosi e di aumento delle cellule cd + non sono costanti bensì possono variare in relazione alla durata e allo stato di attività della malattia. né l'entità della linfocitosi né la percentuale di attivazione di queste cellule hanno valore prognostico o possono orientare il trattamento [ , , ] . il lavaggio broncoalveolare nelle pneumopatie infiltrative diffuse la polmonite da ipersensibilità è caratterizzata dalla presenza di percentuali di linfociti nel bal (spesso > % e talora fino al %) raramente riscontrabili in altre pneumopatie.vi è una netta prevalenza di linfociti cd + con la ratio cd /cd solitamente ridotta (< ). alcuni autori hanno evidenziato casi di polmonite da ipersensibilità con linfocitosi a prevalenza cd e con ratio cd /cd superiore a , [ ] , suggerendo che la ratio cd /cd ha un limitato potere diagnostico. i t linfociti sono a fenotipo cd +/cd +/cd +/cd + [ , , ] . una neutrofilia nel bal è indice di esposizione recente o di malattia in fase avanzata fibrosante [ ] . la presenza di macrofagi schiumosi e mastociti (> %) è un reperto costante (fig. ) [ ] . un simile pattern (alveolite linfocitaria cd +, macrofagi schiumosi, mastociti) si può osservare anche nella tossicità polmonare da farmaci, nella polmonite organizzativa criptogenetica e nella polmonite interstiziale nonspecifica (nsip) (tabelle e ). infine il riscontro di alveolite t linfocitaria con riduzione della ratio cd /cd in un soggetto asintomatico esposto a un possibile antigene scatenante non è da considerare come un indice di malattia bensì solo di esposizione. fig. . sedimento di bal in soggetto affetto da polmonite da ipersensibilità. È presente un cospicuo numero di linfociti con segni di attivazione (nucleo indentato ed ampio citoplasma) accanto ad alcuni macrofagi con citoplasma di aspetto schiumoso (may grunwald giemsa,x ) capitolo endoscopia bronchiale diagnostica dell'adulto la presenza di particelle di polvere nei macrofagi alveolari talora birifrangenti (indice di esposizione ai silicati) o di corpi dell'asbesto (corpi ferruginosi) (fig. ) o la presenza di cellule giganti con segni di cannibalismo (fig. ) (indice di esposizione a metalli duri) nel bal indirizzano verso l'esposizione a polveri, fibre o sostanze inorganiche potenzialmente patogene [ ] . va comunque sottolineato che normalmente l'inquinamento ambientale sottopone qualsiasi individuo ad inalare particelle inorganiche, di cui si può trovare traccia nel bal, e che quindi tali reperti non possono essere diagnostici ma sono solo un segno di avvenuta esposizione. nel bal di pazienti con silicosi semplice si riscontrano un aumento di macrofagi alveolari e linfociti (tabb. e ). nelle forme avanzate con fibrosi massiva si osserva, invece, un aumento di neutrofili polimorfonucleati [ ] . i lavoratori esposti ma non affetti da malattia possono presentare un aumento di linfociti che permette di ipotizzare la presenza di una alveolite subclinica. nei soggetti esposti all'inalazione di amianto il numero di corpi ferruginosi presenti nel bal è proporzionale a quello riscontrabile nei tessuti [ ] . l'analisi mineralogica con microscopia ottica ed elettronica del bal permette di confermare l'avvenuta esposizione o altresì di svelare esposizioni misconosciute [ ] . fig. . sedimento di bal in soggetto esposto ad amianto. in mezzo ad un sedimento cellulare, caratterizzato da macrofagi alveolari con note di antracosi,spicca un corpo dell'asbesto con aspetto biclavato (papanicolau,x ).tale aspetto non è indice di malattia ma solo di esposizione fig. . sedimento di bal in soggetto esposto all'inalazione di metalli duri.nel sedimento accanto ad alcuni macrofagi alveolari si osservano due gigantesche cellule giganti mononucleate con aspetti di cannibalismo (fagocitosi di cellule mononucleate) (may grunwald giemsa,x ) il lavaggio broncoalveolare nelle pneumopatie infiltrative diffuse alveolite emorragica È un reperto comune a diverse patologie (granulomatosi di wegener, poliangite microscopica, sindrome di goodpasture, emosiderosi polmonare idiopatica, connettiviti, reazioni da farmaci etc.) che può essere di entità tale da influenzare l'aspetto macroscopico del bal (recupero francamente ematico) suggerendo la diagnosi ovvero essere diagnosticata anche in caso di sanguinamento occulto. il riscontro, poi, nel bal di macrofagi carichi di emosiderina (siderofagi), indice di sanguinamento verificatosi/insorto da almeno h, permette di discriminare tra sanguinamento dovuto al traumatismo endoscopico e alveolite emorragica vera e propria [ , , ] . nella granulomatosi di wegener gli autoanticorpi c-anca sono dimostrabili nel sovranatante del bal; non è chiaro se possano avere un valore predittivo dell'evoluzione [ ] . nel follow-up dei pazienti con vasculiti o connettiviti il bal viene utilizzato in caso di nuovi infiltrati polmonari per dirimere tra la possibilità di ripresa di malattia, infezione opportunistica o danno da farmaci (ciclofosfamide e metotrexate). la presenza di eosinofilia isolata nel bal superiore al % deve indurre il sospetto di una polmonite eosinofila (acuta o cronica), una sindrome di churg strauss, polmonite eosinofila da farmaci o eosinofilia da parassitosi (tabella ) [ , , ] (fig. ). nel giusto contesto clinico-radiologico, la presenza di un'eosinofilia di tale entità permette, quindi, di porre diagnosi evitando il ricorso alla biopsia polmonare. gli eosinofili sono spesso degranulati ed è frequente osservare macrofagi alveolari in disfacimento. nel sovranatante è possibile evidenziare un forte aumento dell'ecp. la presenza di eosinofilia nel bal (eosinofili superiori al % delle cellule totali) è riportata nel % circa dei pazienti sottoposti a questa procedura per diverse cause; nel % dei casi si tratta di soggetti affetti da pneumopatie infiltrative diffuse, in quest'ambito poi, i livelli di eosinofilia più elevati (> %) sono riscontrati in corso di polmonite eosinofila cronica e nella malattia di churg strauss [ ] [ ] [ ] . molti farmaci possono causare danno polmonare, tra quelli più frequentemente implicati vi sono l'amiodarone, il metotressato ed altri chemioterapici. l'analisi del bal in questi casi può evidenziare la presenza di atipie cellulari, frammenti lipoproteinacei, alveolite linfocitaria, alveolite neutrofila, alveolite eosinofila, alveolite emorragica, aumento dei macrofagi con aspetto schiumoso (tesaurismosi), macrofagi con inclusioni lipidiche [ ] .va comunque sottolineato che uno stesso farmaco può causare diversi tipi di reazione tissutale polmonare, anche in sequenza (tabella ). proprio il metotressato, ad esempio, oltre ad una polmonite interstiziale cronica può causare, anche se meno frequentemente, polmonite organizzativa-bronchiolite obliterante (boop), dad ed edema polmonare. la maggior parte dei pazienti presenta nel bal un'alveolite t linfocitaria ad alta intensità a prevalente fenotipo cd +. sono stati, però, descritti anche casi a prevalente fenotipo cd +. in alcuni casi è stata riportata neutrofilia. il bal è utile soprattutto per escludere forme infettive opportunistiche. la presenza nel sedimento del bal di cellule epiteliali atipiche può rappresentare un segno precoce di evoluzione verso la fibrosi polmonare [ ] . un aspetto comune del bal di pazienti trattati con amiodarone con e senza interessamento polmonare è la presenza di numerosi macrofagi "schiumosi". nei pazienti con lesioni polmonari si osserva invece un'alveolite mista (tabella ) caratterizzata da un aumento dei linfociti, dei neu-il lavaggio broncoalveolare nelle pneumopatie infiltrative diffuse trofili e degli eosinofili. i linfociti risultano essere in prevalenza t cd + (tabella ). tali aspetti possono essere utili alla conferma della diagnosi ma non hanno valore prognostico [ ] . una malattia interstiziale diffusa complica il decorso di una connettivite in circa il % dei casi e ne è spesso un indice prognostico sfavorevole. il tipo di interessamento polmonare è il più vario, spaziando da una polmonite interstiziale (nsip) a quadri di boop, fino a forme di fibrosi interstiziale diffusa, del tutto simili alla fibrosi polmonare idiopatica. il bal riflette questi eventi patologici rivelando un aumento dei linfociti o neutrofili/eosinofili o di ambedue le componenti cellulari (alveolite mista) (tabella ) [ ] . anche in pazienti asintomatici dal punto di vista respiratorio e con lastra del torace normale, il bal può rilevare alterazioni delle componenti cellulari (tabella ). l'importanza prognostica di tale alveolite subclinica in corso di connettivite non è ancora conosciuta [ ] . una alveolite t-linfocitaria subclinica è stata osservata anche in corso di crioglobulinemia mista [ ] . in questo caso l'alveolite subclinica non si è dimostrata predittiva, in un follow-up di anni, di manifesta malattia interstiziale diffusa. l'ards e l'aip sono caratterizzate a livello istopatologico dalla presenza di dad, nell'ards è possibile identificare un evento eziologico scatenante (sepsi, trauma, intervento chirurgico, trasfusioni, etc.), mentre nell'aip no. nella fase precoce dell'ards e dell'aip, il sedimento del bal è caratterizzato da un marcato incremento dei neutrofili (> %) (tabella ), mentre in quella tardiva predominano linfociti ed eosinofili. la persistenza di un elevato numero di neutrofili anche nella fase tardiva è considerato un indice prognostico sfavorevole [ , ] .nel sedimento,si possono osservare anche pneumociti di ii tipo attivati ed aggregati in clusters con atipie morfologiche simil-tumorali (fig. ) [ ] . nel sovranatante sono state riscontrate concentrazioni aumentate di radicali tossici dell'ossigeno, proteasi e citochine (tnf-alpha, il- e ). nei pazienti ricoverati in unità di terapia intensiva per manifestazioni respiratorie, il bal risulta di particolare utilità clinica per differenziare l'ards da: ) alveolite emorragica (emazie e siderofagi); ) polmonite eosinofila acuta (spiccato incremento della popolazione eosinofila); ) polmonite acquisita da ventilatore (presenza di organismi intracellulari [ico] ed esami colturali con carica batterica > cfu/ml); ) neoplasie a rapida progressione come linfangite carcinomatosa, linfoma e leucemia acuta; ) infezioni opportunistiche polmonari (pneumocystis jiroveci, citomegalovirus, aspergillus, etc.) con associato dad [ ] . il fenomeno istopatologico boop non è altro che la fase riparativa successiva a insulti polmonari di varia natura (infettivi, immunologici, tossici). si parla di cop in caso di forma idiopatica. il bal nella boop/cop è caratterizzato da un aumento della cellularità totale con riduzione percentuale della popolazioni macrofagica ed aumento di quella linfocitaria (> %), neutrofila e eosinofila (alveolite mista, tabella ) [ , ] . i linfociti presentano una riduzione della ratio cd /cd , la presenza di un numero elevato di linfociti è un fattore predittivo di buona risposta alla terapia steroidea. sono presenti macrofagi schiumosi e percentuali aumentate di mastociti e plasmacellule [ , ] . il pattern misto (aumento dei linfociti cd +, neutrofili e talora eosinofili) non è specifico e lo si può osservare in corso di alveoliti allergiche estrinseche, polmonite interstiziale nonspecifica e polmonite da farmaci [ ] . circa un % dei casi presenta una linfocitosi con rapporto cd /cd ridotto associata ad aumento dei neutrofili (nsip-cellulata) simile a quello osservabile in corso di boop (tabella ) [ ] . in un'altra metà dei casi è presente un incremento dei neutrofili e degli eosinofili (nsip-fibrotica). queste due alterazioni del sedimento del bal possono essere presenti contemporaneamente. il bal non permette di discriminare tra una fibrosi polmonare idiopatica (uip) e una nsip-fibrotica [ ] . nel bal, nel %- % dei casi, si osserva un aumento delle cellule totali e della percentuale di polimorfonucleati neutrofili (> %) che correlano con l'estensione delle lesioni reticolari in hrtc (tabella ). nel %- % dei casi possono essere aumentati anche i polimorfonucleati eo-sinofili (> %). È inoltre riportato anche un aumento dei linfociti nel %- % dei casi. tale quadro non si differenzia dalla maggior parte delle polmoniti interstiziali idiopatiche o da quello osservabile in altre patologie polmonari fibrosanti (polmoniti da ipersensibilità croniche, nsip fibrotica, asbestosi, etc.) [ , , , ] . un aumento isolato dei linfociti deve far escludere la possibilità di uip. questo valore predittivo negativo del bal è così importante che nelle recenti linee guida congiunte ats ed ers sulla fibrosi polmonare idiopatica il bal viene assunto come uno dei quattro criteri maggiori per porre la diagnosi clinica in assenza di biopsia chirurgica [ ] . il numero od il tipo di cellule del bal non hanno valore prognostico e non sono quindi consigliabili controlli seriati nel tempo per controllare l'evoluzione o la risposta al trattamento [ , ] . durante le fasi accelerate di malattia, dovute al sovrapporsi di un dad, il bal si caratterizza, come nell'aip e nell'ards, per la presenza di marcata neutrofilia (> %) e presenza di pneumociti di ii tipo attivati [ ] . il bal si caratterizza per la presenza di un'alveolite t linfocitaria ad alta intensità a prevalente fenotipo cd + senza caratteri di monoclonalità (tabella ) [ ] . il bal contiene numerosi macrofagi alveolari con inclusioni caratteristiche bronzo-dorate e antracotiche, indistinguibili da quelle che si possono osservare nei fumatori. l'assenza di queste cellule rende la diagnosi di dip improbabile. È segnalato anche un aumento percentuale dei polimorfonucleati neutrofili, degli eosinofili e talora dei linfociti [ , ] . anche se, genericamente, in passato era stato suggerito che una linfocitosi del bal avesse aspetti prognostici positivi (fibrosi polmonare idiopatica e sarcoidosi) [ , , , ] , al momento è ancora aperto il dibattito se il bal sia utile per stabilire l'attività di malattia e abbia quindi valore prognostico nell'ambito delle pneumopatie infiltrative diffuse. allo stesso modo non esistono conferme dell'utilità di bal seriati nel tempo ai fini del monitoraggio della malattia e delle decisioni terapeutiche [ , , , ] . attualmente il bal trova indicazione terapeutica solo nella proteinosi alveolare, quando presente insufficienza respiratoria, al fine di rimuovere meccanicamente il materiale proteinaceo dagli spazi alveolari. generalmente viene lavato un intero polmone attraverso un tubo a doppio lume con il paziente in anestesia generale [ ] . in soggetti che non possono sopportare tale metodica si sono ottenuti buoni risultati anche con bal eseguiti in anestesia locale con quantità totali di - ml, iniettati in varie aliquote, in differenti segmenti e ripetute sedute. bronchoalveolar lavage in interstitial lung disease the role of bronchoalveolar lavage in interstitial lung disease bronchoalveolar lavage and lung histology clinical guidelines and indications for bronchoalveolar lavage:report of european society of pneumology task group on bal technical reccomendations guidelines for bronchoalveolar lavage bronchoalveolar lavage costituents in healthy individuals, idiopathic pulmonary fibrosis,and selected comparison groups bronchoalveolar lavage in the diagnosis of disseminated lung tumors bronchioloalveolar cell carcinoma diagnosed by bronchoalveolar lavage bronchoalveolar lavage in the diagnosis of disseminated lung tumors the value of bronchial washings and bronchoalveolar lavage in the diagnosis of lymphangytic carcinomatosis establishing diagnosis of pulmonary malignant lymphoma by gene rearrangement analysis of lymphocytes in bronchoalveolar lavage fluid bronchoalveolar lavage fluid profiles in sarcoidosis,tuberculosis,non-hodgkin's and hodgkin's disease:an evaluation of differences bronchoalveolar lavage cell data in alveolar proteinosis value of cd- -positive cells in bronchoalveolar lavage fluid for the diagnosis of pulmonary histiocytosis x accumulation of langerhans' cells on the epithelial surface of the lower respiratory tract in normal subjects in association with cigarette smoking diagnosis of pulmonary histiocytosis x by immunodetection of langerhans cells in bronchoalveolar lavage fluid predictive value of bronchoalveolar t cell subset for the course of pulmonary sarcoidosis is the different t helper activity in sarcoidosis and extrinsic allergic alveolitis also reflected by the cellular bronchoalveolar lavage fluid profile? the value of bronchoalveolar lavage in the diagnosis and prognosis of sarcoidosis bronchoalveolar lavage in extrinsic allergic alveolitis:effect of time elapsed since antigen exposure lung t-cells in hypersensitivity pneumonitis:phenotypic and functional analysis mast cells in bronchoalveolar lavage fluid and in transbronchial biopsy specimens of patients with farmer's lung disease inflammation and immune reactions in interstitial lung disease associated with inorganic dust exposure effects of work exposure, retirement, and smoking on bronchoalveolar lavage measurements of lung dust in vermont granite workers diagnostic value of asbestos bodies in broncholaveolar lavage fluid transmission and scanning electron microscopic study of the same cytologic material bronchoalveolar lavage.mosby year book hemosiderin-laden macrophages in bronchoalveolar lavage fluid bronchoalveolar lavage analysis in wegener's granulomatosis.a method to study disease pathogenesis diagnostic significance of increased bronchoalveolar lavage fluid eosinophils analysis of six cases in comparison with other interstitial lung diseases eosinophilic alveolitis in immunologic interstitial lung diseases bronchoalveolar lavage in drug-induced lung disease bronchoalveolar lavage cell profile in methotrexate induced pneumonitis amiodarone pneumonitis bronchoalveolar lavage findings in patients and review of the literature clinical and subclinical alveolitis in connective tissue diseases assessed by bronchoalveolar lavage bronchoalveolar lavage in mixed cryoglobulinaemia associated with hepatitis c virus.british bronchoalveolar lavage in patients with the adult respiratory distress syndrome bronchoalveolar lavage fluid characteristics of early intermediate and late phases of ards hyperplasia of type ii pneumocytes in acute lung injury.cytologic findings of sequential bronchoalveolar lavage bronchoalveolar lavage in intensive care units bronchiolitis obliterans organizing pneumonia (boop): the cytological and immucytological profile of bronchoalveolar lavage bronchoalveolar lavage in bronchiolitis obliterans organizing pneumonia primed by radiation therapy to the breast mast cells in bronchiolitis obliterans organizing pneumonia idiopathic non-specific interstitial pneumonia:comparison with idiopathic pulmonary fibrosis and boop bal findings in idiopathic nonspecific interstitial pneumonia and usual interstitial pneumonia bronchoalveolar lavage in pulmonary fibrosis: comparison of cells obtained with lung biopsy and clinical features idiopathic pulmonary fibrosis:diagnosis and treatment acute exacerbation of idiopathic pulmonary fibrosis: report of a series classification and recent advances in idiopathic interstitial pneumonia long-term durable benefit after whole lung lavage in pulmonary alveolar proteinosis il bal è diventata una procedura diagnostica standard nella maggioranza delle pneumopatie infiltrative diffuse. la tecnica è sicura, minimamente invasiva e in casi selezionati diagnostica (proteinosi alveolare, granulomatosi a cellule di langerhans, infiltrati tumorali e pneumopatie infettive) [ ] [ ] [ ] [ ] [ ] [ ] . in altri casi, il riscontro nella conta cellulare differenziata del bal di una alveolite linfocitaria, neutrofila, eosinofila o mista può fornire dati utili all'orientamento clinico o utili alla diagnosi (tabb. - ) . se, per esempio, i risultati del bal sono compatibili con una determinata diagnosi nel giusto contesto clinico-radiologico (hrct), tale reperto può essere sufficiente a porre la diagnosi. nella diagnostica della fibrosi polmonare idiopatica il bal svolge un ruolo predittivo negativo. il valore clinico della procedura nella stadiazione e nel monitoraggio delle pneumopatia infiltrative è ancora discusso. l'unica indicazione all'impiego del bal ad uso terapeutico è la proteinosi alveolare. key: cord- -zpblaa authors: buchan, blake w.; windham, sam; balada-llasat, joan-miquel; leber, amy; harrington, amanda; relich, ryan; murphy, caitlin; dien bard, jennifer; naccache, samia; ronen, shira; hopp, amanda; mahmutoglu, derya; faron, matthew l.; ledeboer, nathan a.; carroll, amanda; stone, hannah; akerele, oluseun; everhart, kathy; bonwit, andrew; kwong, christina; buckner, rebecca; warren, del; fowler, randal; chandrasekaran, sukantha; huse, holly; campeau, shelley; humphries, romney; graue, corrin; huang, angela title: practical comparison of the biofire filmarray pneumonia panel to routine diagnostic methods and potential impact on antimicrobial stewardship in adult hospitalized patients with lower respiratory tract infections date: - - journal: j clin microbiol doi: . /jcm. - sha: doc_id: cord_uid: zpblaa lower respiratory tract infections, including hospital-acquired and ventilator-associated pneumonia, are common in hospitalized patient populations. standard methods frequently fail to identify the infectious etiology due to the polymicrobial nature of respiratory specimens and the necessity of ordering specific tests to identify viral agents. the potential severity of these infections combined with a failure to clearly identify the causative pathogen results in administration of empirical antibiotic agents based on clinical presentation and other risk factors. we examined the impact of the multiplexed, semiquantitative biofire filmarray pneumonia panel (pn panel) test on laboratory reporting for adult inpatients submitting bronchoalveolar lavage (bal) specimens for laboratory analysis. the pn panel demonstrated a combined . % positive percent agreement (ppa) and . % negative percent agreement (npa) for the qualitative identification of bacterial targets compared to routine bacterial culture. semiquantitative values reported by the pn panel were frequently higher than values reported by culture, resulting in semiquantitative agreement (within the same log( ) value) of . % between the pn panel and culture; however, all bacterial targets reported as > ( ) cfu/ml in culture were reported as ≥ ( ) genomic copies/ml by the pn panel. viral targets were identified by the pn panel in . % of specimens tested, of which . % were detected in conjunction with a bacterial target. a review of patient medical records, including clinically prescribed antibiotics, revealed the potential for antibiotic adjustment in . % of patients based on the pn panel result, including discontinuation or de-escalation in . % of patients, resulting in an average savings of . antibiotic days/patient. l ower respiratory tract infections, including community-acquired pneumonia (cap), hospital-acquired pneumonia (hap), and ventilator-associated pneumonia (vap), are linked to significant morbidity and mortality ( ) ( ) ( ) ( ) . these infections can be caused by bacterial, viral, or fungal agents, depending on patient exposure and clinical risk factors. bacterial pathogens associated with hap and vap, including staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, and other members of the enterobacteriaceae, are often multidrug resistant ( ) . this includes carbapenemaseproducing organisms, which have been independently associated with higher mortality rates following infection ( ) . selected studies have identified a significant decrease in mortality for patients who receive prompt and effective antibiotic therapy to treat pneumonia ( , , ) . based on these findings, broad-spectrum empirical antibiotic therapy consisting of or agents is recommended for patients with symptoms consistent with hap or vap until definitive laboratory results are available to inform targeted and specific therapy ( , ) . as a result, it is estimated that up to % of antibiotics used in hospital intensive care units (icus) are prescribed to treat these infections ( , ) . an emphasis of the current american thoracic society (ats) and infectious disease society of america (idsa) guidelines for management of patients with hap or vap is to reduce exposure to broad-spectrum and unnecessary antibiotics by targeting therapy to treat the most likely pathogens based on patient risk factors and laboratory results ( ) . laboratory identification of a specific infectious etiology in patients with pneumonia has been associated with a statistically significant reduction in mortality, presumably because it enables targeted effective therapy ( ) . historically, quantitative and qualitative bacterial culture has been the primary approach for laboratory diagnosis of lower respiratory tract infections, including pneumonia. these methods are useful in establishing definitive antibiotic therapy; however, recovery of potential pathogens is variable due to antibiotic exposure prior to specimen collection, fastidious growth characteristics of some pathogens, or overgrowth of resident flora ( ) . these factors contribute to variable culture sensitivity and turnaround times of h or more. furthermore, additional specific culture or molecular tests are required to identify atypical bacteria or viral pathogens, and these tests may not be routinely ordered by clinicians. combined, these shortcomings diminish the ability of current standard-of-care (soc) methods to rapidly and accurately identify specific infectious etiologies and target antibiotic therapy for these infections. molecular diagnostics, including pcr-based tests, generate a sensitive result within hours of specimen collection. these tests have the potential to reduce the duration of broad-spectrum empirical antibiotic therapy by identifying pathogenic organisms or specific antibiotic resistance markers to days sooner than routine methods. specifically, rapid molecular detection of methicillin-resistant s. aureus (mrsa) in respiratory specimens has been associated with a potential % to % reduction in anti-mrsa antibiotic days and a total savings of Ͼ$ , in antibiotic cost annually ( ) . multiplexed molecular tests, including the eplex rp (genmark diagnostics) and the biofire filmarray respiratory (rp ) panel (biofire diagnostics), enable broad detection of viral pathogens in respiratory specimens but identify only a few bacteria, including mycoplasma pneumoniae, chlamydia pneumoniae, and bordetella species ( ) . the filmarray pneumonia panel (pn panel; biofire diagnostics, llc) is a sample-toanswer pcr-based in vitro diagnostic test that analyzes native (untreated) sputum (including endotracheal aspirates) and bronchoalveolar lavage (bal) (including mini-bal) specimens for the presence of bacteria, viruses, and genetic markers of antimicrobial resistance within approximately min, with Ͻ min of hands-on time. the pn panel reports qualitative ("detected" or "not detected") results for viral targets, bacterial targets associated with atypical pneumonia, and antibiotic resistance markers while providing a semiquantitative value for additional bacterial targets commonly associated with respiratory infections (table ) . semiquantitative reporting is intended to facilitate interpretation of results based on the absolute and relative abundance of each target detected. this is consistent with current culture-based laboratory protocols for analysis and reporting of respiratory specimens proposed by the idsa and others to aid in discrimination of true infection from clinically insignificant colonization of the airway ( , , ) . these culture-based guidelines propose specimen-specific thresholds ranging from cfu/ml for bal specimens to to cfu/ml for endotracheal aspirates (eta) and sputa to define clinically significant infection and minimize reporting of low-abundance, likely commensal organisms to reduce the use of potentially unnecessary antibiotics. similarly, the pn panel also has a detection threshold intended to prevent reporting of bacteria present at low levels in respiratory specimens. the analytical and clinical performance (accuracy) of the pn panel was established based on data collected from over , clinical bal and sputum specimens collected across u.s. medical centers during the clinical evaluation that was performed for regulatory clearance ( ) . the aim of this study was to conduct a practical analysis of a subset of those specimens comparing results reported using routine soc methods to those obtained using the pn panel and to assess the potential impact of the pn panel results on antibiotic utilization in these patients. specifically, we compared positivity rates in terms of total positive specimens and total targets detected using soc versus the pn panel, as well as the absolute and relative concordance of quantitative results for identified bacterial targets. we also noted the incidence of viral targets detected by the pn panel compared to clinician-ordered soc viral tests. finally, we conducted a retrospective chart review to assess the potential impact of the pn panel results on antibiotic modifications and stewardship. clinical specimens (bronchoalveolar lavage [bal], mini-bal, and endotracheal aspirate [eta] specimens and sputum from subjects of all ages and multiple care settings) were enrolled at eight u.s. clinical centers between october and july as part of the clinical trial for regulatory clearance of the pn panel ( ) . for this study, subsets of the clinical trial specimens comprising residual bal (n ϭ ) or mini-bal (n ϭ ) specimens were selected for further analysis. bal is a bronchoscopy-guided procedure which installs to ml of saline for specimen collection, whereas mini-bal uses a nonbronchoscopy-guided "blind" lavage catheter and installs approximately ml of saline into the lung. specimens were specifically selected from adult inpatients, since this population is at highest risk for hap and vap and frequently receives broad-spectrum empirical antibiotic therapy for presumptive respiratory tract infections, including those caused by multidrug-resistant pathogens. sputum and endotracheal aspirates were specifically excluded from the analysis. based on the full clinical trial set, sputum and endotracheal aspirate specimens were over three times as likely as bal or mini-bal specimens to have Ն targets detected ( bal and sputum specimens) and almost twice as likely to have bacterial targets that were not recovered by specialized reference culture methods ( bal and sputum specimens) ( ) . as a result of this added complexity, we chose to focus on the analysis of bal and mini-bal specimens in the current study. the final cohort included randomly selected specimens from each study site proportional to the total specimens enrolled at that site. this included specimens submitted to the medical college of wisconsin, milwaukee, wi (n ϭ ); the ohio state university wexner medical center, columbus, oh (n ϭ ); nationwide children's hospital, columbus, oh (n ϭ ); loyola university medical center, maywood, il (n ϭ ); indiana university school of medicine, indianapolis, in (n ϭ ); university of nebraska medical center, omaha, ne (n ϭ ); and university of california los angeles health, los angeles, ca (n ϭ ). the pn panel result was compared to the soc result(s) reported by each clinical laboratory to assess the impact of the pn panel on result reporting, positivity, and the potential for antibiotic stewardship. the average age of enrolled patients was . years (range, to years), and . % were male. all soc and pn panel testing was initiated within h of collection at the study enrollment site. each clinical site followed its own routine procedures for defining specimen acceptability criteria, selection of culture medium, culture workup, bacterial identification, susceptibility testing, and result reporting (site-specific methods are detailed below). chart review and data abstraction. clinical, demographic, and laboratory data were abstracted from the laboratory information system (lis) and electronic health record (ehr) at each clinical site by individuals who were not involved in either clinical testing or pn panel testing of specimens and who were blinded to pn panel results. the lis was reviewed to identify clinician-ordered tests and soc results associated with each submitted specimen, including bacterial culture, antimicrobial susceptibility tests (asts), and molecular tests for viral agents that were ordered as part of clinical care. soc testing results encompassed all laboratory results reported from the same specimen tested with the pn panel, as well as other respiratory specimens collected within Ϯ h of the enrolled specimen to provide a comprehensive comparison. the ehr was reviewed to retrieve clinical and demographic data, including hospitalization status at the time of specimen collection, subject age, and subject gender, and to ascertain antibiotic use from days preceding to days following specimen collection. abstracted lis and ehr data were used to assess antibiotic adjustments based on soc results and to determine the potential impact of the pn panel results according to criteria described below. a waiver of the requirement for informed consent was obtained from the institutional review board (irb) at each study site for the use of residual specimens and for the abstraction and deidentification of subject information from the laboratory and medical record. biofire pn panel. testing of specimens using the pn panel was conducted at each site in accordance with the manufacturer's instructions for use and site-specific laboratory protocols to ensure safe handling of specimens and maintenance of quality. all primary specimens were handled within in a biological safety cabinet (bsc) during processing for pn panel testing. a flocked swab (provided) was used to sample each bal specimen and inoculate a filmarray injection vial (faiv) containing sample buffer. inoculated faivs were capped and inverted three times to facilitate organism release, and contents were then injected into the pn panel test pouch. inoculated pouches were inserted into the filmarray instrument for analysis (ϳ -min run time). each specimen was processed separately, and the bsc was surface disinfected prior to processing subsequent specimens. the pn panel test pouch contains all reagents necessary for specimen lysis, nucleic acid extraction, reverse transcription, amplification, and detection of genomic sequences unique to each of the panel targets (table ). in addition, the test pouch contains two internal controls to assess pouch function and enable the calculation of semiquantitative results. if either internal control fails, a result of "invalid" is reported. for viral and atypical bacterial targets, a qualitative result of either "detected" or "not detected" is reported. for bacterial targets reported semiquantitatively, the pn panel reports the specific target with a log binned value of , , , or Ͼ genomic copies/ml; targets quantified by the internal software at . to . copies/ml are reported as copies/ml; targets quantified at . to . copies/ml are reported as copies/ml, etc. targets quantified at Ͻ . copies/ml are reported as "not detected." genetic markers of antimicrobial resistance are reported qualitatively as "detected" or "not detected" only if a commensurate bacterial target is detected and reported (table ) . standard-of-care testing. standard-of-care testing at each of the seven clinical centers was conducted in accordance with clinician test orders and local laboratory protocols. bacterial culture was conducted by inoculating a portion of the bal fluid into several selective and differential growth media, including blood agar, chocolate agar, macconkey agar, columbia colistin-nalidixic acid (cna) agar, and haemophilus isolation agar, in accordance with site-specific protocols. plates were inoculated and streaked using calibrated loops in accordance with local protocols to achieve a semiquantitative culture result. inoculated media were incubated at °c in a % co atmosphere and were examined daily for bacterial growth. bacterial colonies were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof ms) as the primary method, supplemented with a combination of manual biochemical tests and automated phenotypic identification systems when necessary in accordance with local protocols. potential pathogens and normal flora were reported semiquantitatively in cfu per milliliter by of laboratories; a single laboratory reported semiquantitative results using descriptors (e.g., "rare," "few," "moderate," or "many"). potential pathogens quantified at Ͻ cfu/ml (or "rare") were not routinely reported unless they were in pure culture or were associated with polymorphonuclear leukocytes (pmns) in the primary gram stain. antibiotic susceptibility tests (asts) were conducted using automated ast instruments, including the vitek- (biomérieux, durham, nc) and bd phoenix (bd, sparks, md). additional phenotypic tests, including disk diffusion or etest (biomérieux, durham, nc), were used when necessary (e.g., when automated ast failed or to determine the presence of extended-spectrum beta-lactamase) in accordance with local protocols. molecular tests for viral pathogens were conducted using fda-cleared (genmark esensor xt- , luminex verigene rpp, or the biofire filmarray respiratory [rp] panel) and laboratory-developed assays at each site upon clinical order. assessment of the potential impact of pn panel results on antibiotic therapy and stewardship. the potential impact of pn panel results on antibiotic therapy and stewardship was assessed based on a comparison of the pn panel results to the results of the clinician-ordered soc tests performed on each bal or mini-bal specimen. it was assumed that all patients with a clinical order for microbiologic testing on a bal or mini-bal specimen were being evaluated for diagnosis of pneumonia and that antimicrobials were not being used to treat any concomitant infection unless otherwise indicated by chart review. antimicrobials targeting anaerobic pathogens (e.g., metronidazole) and those not used for treatment of pneumonia (e.g., daptomycin) were not included in the analysis. soc bal specimen culture results were considered the gold standard and were used to define potential modifications based on the pn panel result as inappropriate or appropriate. if a diagnostic test was not performed as part of soc, but a potential pathogen was detected by the pn panel (e.g., the pn panel was positive for influenza virus but no soc pcr for influenza virus was performed), the pn panel result was considered correct. this rationale was used based on the observed Ͼ % sensitivity and Ͼ % specificity of the pn panel for identification of viral targets in bal specimens compared to conventional pcr tests (followed by sequencing) for each analyte ( ) . potential therapy modifications were classified as (i) appropriate antibiotic escalation, (ii) appropriate antibiotic de-escalation, (iii) inappropriate antibiotic escalation or continuation, (iv) inappropriate antibiotic de-escalation, or (v) no change, in accordance with definitions and examples provided in table s . antibiotic escalations were defined as any broadening of antimicrobial spectrum, which could include a change in agent (e.g., ceftriaxone to cefepime) or the addition of an agent(s). antibiotic deescalation was defined as any narrowing of antimicrobial spectrum, which could include a change in agent (e.g., cefepime to ceftriaxone) or discontinuation of an agent(s) in a multidrug regimen. it was possible for a single patient to qualify for more than one potential intervention, depending on the number of organisms identified, specific organism identification, and empirical antibiotic therapy regimen. the time of specimen collection, time of final culture result, and time(s) of antibiotic initiation or discontinuation were collected and used to categorize the potential antibiotic modifications. the time of the pn panel result was set as h from the time of specimen collection, based on a combined estimate of specimen transport time, in-lab processing, and pn panel run time (approximately min). if empirical antibiotics were initiated, it was assumed that these could be discontinued based on concordant negative pn panel and culture results as soon as the pn panel result was available. if both pn panel and soc results yielded the same clinically relevant organism(s), it was assumed that antibiotics could be initiated, escalated, or de-escalated to optimal therapy as soon as pn panel results became available. the number of hours of antibiotic saved or hours earlier that antibiotics could have been initiated or escalated was calculated based on the difference between the time of pn panel result (i.e., h after specimen collection) and the actual time of antibiotic change, which was obtained from chart review and was assumed to be based on the soc culture and/or pcr result(s). if discontinuation times for antibiotics were not found in chart review, they were still included in the analysis of potential escalation or de-escalation but were excluded when the number of antibiotic hours saved or number of hours sooner that appropriate antibiotics could have been initiated was calculated. statistical analysis. clinical performance of the pn panel, including sensitivity and specificity, was calculated using standard methods, including binomial two-sided % confidence intervals ( % ci), which were calculated in accordance with methods described by newcombe ( ) . impact of the pn panel on overall specimen and target positivity. bacterial culture was ordered for all bal specimens evaluated in this study. among these, ( . %) specimens were reported as positive for at least one on-panel bacterial target by both culture and the pn panel. an additional ( . %) specimens were reported as positive for at least one bacterial target by the pn panel but had culture results reported as "negative/no growth" ( / ; . %) or "negative/normal oral flora" ( / ; . %) by the reporting laboratory (fig. a ). across all specimens tested, a total of individual bacterial targets were detected by both the pn panel and culture. an additional targets were detected by the pn panel alone, and on-panel targets were detected by culture alone (fig. b) . these data demonstrate a . % increase in the number of bal specimens reported as positive and a . % increase in individual on-panel targets reported when the pn panel was compared to routine bacterial culture. off-panel bacterial targets were reported by culture in ( . %) specimens. these included haemophilus parainfluenzae (n ϭ ), burkholderia cepacia (n ϭ ), morganella morganii (n ϭ ), providencia stuartii (n ϭ ), lactobacillus sp. (n ϭ ), corynebacterium spp. (n ϭ ), achromobacter spp. (n ϭ ), viridans group streptococci (n ϭ ), enterococcus spp. (n ϭ ), "beta-hemolytic streptococcus not type a" (n ϭ ), coagulasenegative staphylococci (n ϭ ), and other bacteria that were not definitively identified by the reporting clinical laboratory (n ϭ ). many of these targets are not considered respiratory pathogens unless they are in pure cultures or predominant; however, / ( . %) were quantified at Ն cfu/ml and may have been clinically significant. molecular tests for viral pathogens were clinically ordered for only / ( . %) bal specimens submitted for bacterial culture and included primarily multiplexed respiratory panel tests (n ϭ ) and influenza virus or respiratory syncytial virus (rsv) tests (n ϭ ) ( fig. a) . the soc positivity rate was / ( . %) among specimens with a clinical order for viral-pathogen testing. at least one viral target was detected by the pn panel in / ( . %) bal specimens. among these, / ( . %) were also positive for at least one bacterial target by the pn panel (fig. b ). only / ( . %) specimens with a positive viral detection by the pn panel had a clinician-ordered molecular test for viral pathogens, demonstrating that a large proportion of viral targets go undetected due to a lack of corresponding test orders. qualitative agreement of the pn panel with routine culture for bacterial targets. an assessment of the overall qualitative performance of the pn panel for detection of bacterial targets demonstrated . % ( / ) positive percent agreement (ppa) and . % negative percent agreement (npa) ( , / , ) with routine culture results (table ) . notably, these data are similar to the sensitivity reported for bal specimens compared to an expanded quantitative reference culture method ( . %) and reported soc results ( . %) in the clinical trial study ( ) . all culture-positive, pn panel-negative targets were detected at a low quantity in the bal specimens. they included (i) e. coli reported at cfu/ml in a culture also containing s. aureus at cfu/ml and k. pneumoniae at cfu/ml (both s. aureus and k. pneumoniae were detected by the pn panel), (ii) s. aureus reported at cfu/ml in culture (the pn panel detected streptococcus agalactiae), and (iii) p. aeruginosa reported as "few" in culture (the pn panel was negative for all targets). it is possible that each of these organisms was present at a concentration below the pn panel threshold for reporting a positive result, which was specifically designed to be . genomic copies/ml in accordance with current guidelines to reduce the risk of reporting clinically insignificant results in bal specimens ( , ) . laboratories adhering to these guidelines may not routinely report the presence organisms at this quantity in cultured bal specimens. the pn panel identified bacterial targets that were not reported by routine culture. the most common culture-negative detections were s. aureus (n ϭ ) and h. influenzae (n ϭ ), though at least one culture-negative detection was observed for all but two of the pn panel bacterial targets ( table ) . a chart review revealed that ( . %) of these culture-negative detections were made in specimens obtained from patients that had received at least one dose of an antibiotic with potential activity against the specific bacterial target within the h preceding specimen collection (fig. ). an additional ( . %) culture-negative detections had a routine culture report indicating the presence of "normal oral flora," which could have obscured detection and reporting of these bacterial targets in culture. furthermore, ( . %) of these detections were quantified at genomic copies/ml by the pn panel, indicating a low concentration of target that may have been overlooked or disregarded during routine culture workup of specimens containing significant normal oral flora. the remaining ( . %) culture-negative detections were in samples from patients without antibiotic exposure and in cultures without a report of "normal oral flora." in / ( %), the target was quantified as genomic copies/ml by the pn panel and may have been below the culture threshold for detection or reporting. the final culture-negative detections were reported at or genomic copies/ml by the pn panel. semiquantitative agreement of the pn panel bacterial targets with routine culture. semiquantitative results of the pn panel (number of genomic copies per pn panel) . the culture-negative targets detected by the pn panel were reported at concentrations ranging from to Ն genomic copies/ml. nearly % ( / ) of these detections were in specimens obtained from patients who had received potentially effective antibiotic therapy within h preceding specimen collection. correlation of the pn panel and culture in identifying the predominant bacterial target in bal specimens. in addition to semiquantitative concordance, we investigated the concordance of results based on relative abundance of targets detected by the pn panel and culture (i.e., rank order of targets detected in a specimen). at least one bacterial target was detected by both the pn panel and routine culture in specimens, including specimens with multiple bacterial targets ( to ) reported by the pn panel and/or culture. the overall concordance between the pn panel and culture for reporting the same bacterial target as most predominant in a given specimen was . % ( / ) ( table ). this included % concordance for all specimens with a single target detected by both the pn panel and culture and . % ( / ) for specimens with multiple targets detected by the pn panel and/or culture. two of the four discordant results occurred in specimens with one target reported by culture and two reported by the pn panel. in both cases, the pn panel reported s. aureus as the predominant target at a concentration log higher than enterobacter cloacae, whereas culture reported only the e. cloacae (i.e., no s. aureus present). a chart review revealed that both patients had received antistaphylococcal antibiotics within h preceding specimen collection, which may have accounted for the culture-negative result. a third discordant result occurred in a specimen that was reported as positive for streptococcus pneumoniae and p. aeruginosa by both the pn panel and culture; however, the pn panel reported s. pneumoniae as predominant (s. pneumoniae, copies/ml; p. aeruginosa, copies/ a numbers in parentheses are the numbers of culture-negative results obtained for specimens from patients who received antibiotics with potential activity against the given bacterial target detected within h preceding specimen collection. one laboratory reported bacterial culture quantitation ( isolates) as "few," "moderate," or "many"; these were categorized as , , and Ն cfu/ml, respectively. shading indicates concordance between the biofire pn panel and routine culture quantitation. b concordance between the pn panel and culture quantitation among all positive cultures was . % ( / ). ml), while culture reported p. aeruginosa as predominant (p. aeruginosa, cfu/ml; s. pneumoniae, "few"). this patient had no record of receiving antibiotics prior to specimen collection. the discordance in predominant target may be due to a difference in growth rate, poor recovery of these bacteria in culture, or inaccurate molecular quantitation by the pn panel. notably, analytic studies assessing the accuracy of the pn panel quantitation using quantified reference standards demonstrated . % accuracy within the defined log bins, suggesting that this discordance was likely related to the fastidious growth characteristics of s. pneumoniae and failure to accurately quantify in culture ( ) . the final discordant result occurred in a specimen that was reported as positive for klebsiella oxytoca, serratia marcescens, p. aeruginosa, and s. aureus by both the pn panel and culture. k. oxytoca, s. marcescens, and p. aeruginosa were all quantified at equivalent copies or cfu/ml by the pn panel and culture, respectively; however, s. aureus was reported as predominant by the pn panel ( copies/ml), whereas culture reported s. aureus as the least abundant target in the specimen ( cfu/ml). this patient had no record of receiving antibiotics prior to specimen collection. detection of markers of antibiotic resistance. the presence of genetic resistance markers is reported conditionally by the pn panel, only for specimens with a commensurate bacterial pathogen detected (table ) ( ) . this is similar to the reported sensitivity ( % to %) and specificity ( % to %) of molecular tests designed to specifically detect mrsa in nasal specimens, as well as results obtained using these tests to detect mrsa in bal specimens ( ) ( ) ( ) . the pn panel reported the presence of a carbapenemase gene (bla kpc , bla ndm , bla vim , bla imp , or bla oxa- ) or an extended-spectrum beta-lactamase (esbl) gene qualitative agreement of viral targets with standard-of-care methods. among bal specimens tested, the pn panel detected a total of viral targets in unique specimens ( table ). the most commonly detected target was human rhinovirus/ enterovirus (n ϭ ), followed by coronavirus (n ϭ ) and influenza a virus (n ϭ ). a soc test for viral agents was ordered for only / ( . %) specimens. among these, the pn panel demonstrated . % ( / ) agreement with the soc result, with no false-positive detections. three discordant results included one specimen reported as positive for parainfluenza virus by the biofire rp panel, one specimen positive for influenza a virus by the xpert flu/rsv xc assay (cepheid, sunnyvale, ca), and one specimen positive for adenovirus by the genmark xt- respiratory viral panel that were reported as "not detected" by the pn panel. of note, soc data were abstracted from the medical record to include all test results reported within h of the specimen's being tested by the pn panel. therefore, it is possible that the discordant results were obtained from soc testing of a different specimen or specimen type than that tested by the pn panel (e.g., a nasopharyngeal swab collected the same day as the bal specimen). importantly, only . % ( / ) of all specimens with a positive pn panel result had a standard-of-care order for viral pathogen testing. the lack of soc comparator test orders for the majority of specimens enrolled limits the ability to assess sensitivity and specificity of the pn panel for viral targets. however, it highlights an apparent underappreciation of the prevalence of viral pathogens in this patient population, which may result in delayed or missed diagnosis, missed opportunity to de-escalate antibiotics, and missed opportunity for the implementation of appropriate infection prevention strategies within the hospital icu setting. specifically, only / specimens testing potential impact of the biofire pn panel on antibiotic utilization. complete medical chart data were available for / ( . %) patients whose specimens were included in this study. potential antibiotic adjustments were based on comparison between the pn panel and routine culture results. actual antibiotic prescription and modification based on the soc results were determined by medical chart review. potential antibiotic adjustments based on the pn panel results were considered appropriate only if pn panel and soc results were in positive or negative agreement. in these cases, it was assumed that the same appropriate adjustment could have been made at the time of pn panel result. potential antibiotic adjustments based on pn panel results were considered "inappropriate" if results between pn panel and soc were discordant (see table s and materials and methods for specific criteria). there was a potential for antimicrobial modification based on pn panel results for / ( . %) of evaluable patients (table ) . for many patients, there was an opportunity for multiple antimicrobial modifications, including simultaneous escalation and de-escalation, due to the empirical utilization of multiple antibiotics in a single patient (average of . potential modifications per patient). the most common potential intervention was an appropriate antibiotic deescalation or discontinuation, which encompassed total antimicrobials ( . % of all antimicrobials considered) in individual patients ( . % of all patients considered). these de-escalations and discontinuations resulted in a potential to reduce the duration of unnecessary antibiotic therapy by over , cumulative hours in these patients. on a per-patient basis, this equated to an average potential of . fewer total antibiotic days per patient or . days per antibiotic. pn panel results most commonly allowed de-escalation or discontinuation of vancomycin ( % of de-escalations/discontinuations) and piperacillin-tazobactam ( % of de-escalations/discontinuations) due to negative results for mrsa and gram-negative bacilli, respectively (fig. ) . appropriate escalation or initiation of antibiotics would have been possible in / ( . %) of patients based on positive agreement between pn panel and soc results, enabling initiation of active antibiotics a combined total of . h earlier than routine culture results for these patients. specifically, for of the patients, pn panel results would have resulted in reduced time to appropriate gram-negative coverage for patients with pneumonia secondary to gram-negative organisms inadequately covered by the empirical antibiotic regimen. in / patients, pn panel results would have prompted mrsa coverage to be initiated sooner. finally, in / patients, the pn panel detected influenza a or b virus when no clinician order for an influenza virus test had been placed. this would have enabled rapid initiation of antiviral therapy as well as droplet isolation, neither of which was implemented for these patients. the pn panel result might have alternatively prompted inappropriate antimicrobial de-escalation or discontinuation in four patients. for three of these patients, the pn panel failed to detect an organism isolated by routine culture methods. of note, in all cases, the quantity of the organism reported in routine culture was below the threshold for clinical significance recommended by current guidelines ( cfu/ml or "few") ( table ) and was likely below the designed lower limit of detection of the pn panel. despite this, it is possible that acting upon the negative pn panel result would have resulted in inappropriate de-escalation of antimicrobials and potential undertreatment if the organisms were of clinical relevance. for the remaining patient, routine culture yielded . ϫ cfu/ml methicillin-resistant s. aureus (mrsa). the pn panel identified s. aureus ( copies/ml) but failed to detect meca/mecc and mrej. there were culture-negative specimens with at least one bacterial target detected by the pn panel ( with single targets and with multiple targets). in the majority of these cases ( / ; . %), soc reported "normal oral flora." due to laboratory-specific culture reporting protocols, identification of individual bacteria present in these specimens was not completed. furthermore, / ( . %) targets were quantified at copies/ml by the pn panel, indicating a low organism burden. in these specimens, utilization of the pn panel result might have resulted in inappropriate initiation or escalation/continuation of antimicrobials that were not prescribed based on culture results. finally, for / ( . %) of patients, pn panel and soc were in either positive or negative agreement and the patient was receiving antibiotic therapy at the time of sample collection that was determined to be optimal for the identified organisms. therefore, no antibiotic modification would have been made based on the pn panel result. this study was specifically designed to provide a pragmatic assessment of the pn panel, including a comparison to routine laboratory test results and the potential impact of results on antibiotic therapy. as such, pn panel results were compared to the standard-of-care method(s) employed at each participating laboratory rather than a standardized culture protocol. this included a comparison to any culture-based or molecular tests ordered for clinical care within h preceding to h following collection of the specimen tested with the pn panel. no additional tests were conducted on specimens to resolve discrepant results or provide a comparator if a clinical test order was not received (e.g., the pn panel detected influenza a virus but no soc test for respiratory viruses was ordered). a comprehensive analytic evaluation of the pn panel study is provided by murphy et al. ( ) . compared to soc bacterial culture methods, use of the pn panel resulted in a . % increase in specimens reported as positive and a . % increase in the total number of bacterial targets detected. these specimens were reported as "negative/no growth" ( . %) or "negative/normal oral flora" ( . %) based on routine culture and reporting protocols. we did not perform additional tests to confirm the accuracy of pn panel results in culture-negative specimens; however, Ͼ % of culture-negative discordant results were found to be positive using an alternative molecular test or were below the culture threshold for reporting during the clinical trial for regulatory clearance, suggesting that these are not false-positive detections ( ) . in addition, our results are comparable to findings published by lee et al., who reported a . % increase in total bacterial targets detected by the pn panel among bal and endotracheal aspirate specimens ( ) , and ozongwu et al., who reported a . % increase in positive specimens and a . % increase in total bacterial targets detected using different multiplex molecular assays ( ) . the increase in positive results reported by molecular tests is not unexpected due to the detection of both viable and nonviable organisms, as well as detection of low-abundance targets and those not recovered in culture due to fastidious growth characteristics. however, the interpretation and potential significance of these results require special attention to determine the impact of reporting on patient management. in our study, approximately one-quarter ( / ; . %) of the culture-negative detections were quantified as copies/ml by the pn panel. semiquantitative values reported (in copies per milliliter) by the pn panel are on average approximately log higher than values (cfu per milliliter) reported from culture. this quantitative discordance was noted in our study (discussed below) as well as in other preliminary evaluations of the pn panel ( , , ) . therefore, targets quantified as copies/ml by the pn panel may frequently be quantified as cfu/ml by routine culture. this would approach the culture-based limit of detection of cfu/ml if one is relying on the use of a -l loop for specimen inoculation. furthermore, potential pathogens present at Ͻ cfu/ml in culture may not be routinely reported in accordance with current guidelines ( , ) . studies comparing the clinical outcomes of patients stratified by culture-based quantification of bacterial targets have demonstrated no increase in mortality for patients with bacteria quantified below cfu/ml in bal specimens ( ) . the clinical significance of additional culture-negative low-concentration ( copies/ ml) detections by the pn panel requires further investigation, especially given that these detections have historically gone largely unreported based on current culture and reporting guidelines. in the absence of these studies, such results should be interpreted with caution and in the context of other laboratory results (e.g., additional pathogens present at high concentrations in the same specimen) and clinical symptoms. another potential factor resulting in culture-negative, pn panel-positive results is the use of empirical antibiotics prior to specimen collection. exposure to antibiotic therapy for as little as h prior to collection can dramatically reduce culture-based recovery of potential pathogens from clinical specimens ( ) ( ) ( ) . up to % of pcrpositive, culture-negative results may be attributable to recent exposure to empirical antibiotics, underscoring both the frequency of empirical antibiotic use in these patients and the negative consequence for culture-based diagnosis ( , ) . in our study, nearly % ( / ) of culture-negative detections by the pn panel were in specimens obtained from patients who received antibiotic therapy within h preceding specimen collection. the pn panel quantitation of these targets ranged from to Ͼ copies/ml, including / ( . %) reported at Ն copies/ml. these notably included s. aureus (n ϭ ), h. influenzae (n ϭ ), s. pneumoniae (n ϭ ), and p. aeruginosa (n ϭ ), among others. unlike culture-negative targets quantified as copies/ml, these may be more likely to represent clinically significant infections and would have been more likely to be recovered and reported by routine culture had the patient not received preemptive antibiotics. the potential benefit of these pn panel detections is -fold. first, detection of a high-concentration (clinically significant) pathogen could potentially prevent the early termination of effective antibiotics. current guidelines recommend a -day antibiotic course for treatment of hap and vap ( ) . a negative culture result at h may result in premature discontinuation and risk of relapse. second, identification of a specific pathogen(s) could enable modification (escalation or de-escalation) of empirical antibiotic therapy even in the face of a negative culture. for example, a pn panel result of h. influenzae or s. pneumoniae could enable de-escalation of empirical broad-spectrum agents, such as vancomycin and cefepime or meropenem, to a more narrow-spectrum beta-lactam regimen, such as ceftriaxone or amoxicillin. appropriate narrowing or discontinuation of antibiotic therapy based on quantitative culture results has been associated with a decrease in subsequent infection with multidrug-resistant organisms (mdros) ( ) . early identification of bacterial pathogens by the pn panel, even in negative cultures, may have a similar impact. confirmation of these hypotheses would require a longitudinal comparison of patients whose antibiotics were withheld, modified, or continued broadly based on a positive pn panel but negative culture result. unfortunately, the design of our study did not allow the collection of these data, and this remains an area of significant interest requiring research. quantitative reporting of bacterial counts in bal specimens has been recommended to aid in the interpretation of results ( ) . the importance of quantitative molecular reporting for bacterial pathogens in respiratory specimens was demonstrated by a recent study evaluating a multiplex molecular test that reports only qualitative results. compared to quantitative s next-generation sequencing (ngs) analysis, targets identified by the qualitative test spanned a wide range from . % to . % of all ngs reads obtained from the specimen ( ) . targets with a low percentage of reads frequently were not recovered in culture, leading to better agreement between the quantitative molecular approach (ngs) and culture. detection of low-abundance organisms or an inability to differentiate predominant organisms in polymicrobial cultures may limit the ability to appropriately modify antibiotic therapy or result in treatment of clinically insignificant colonizing bacteria ( , ) . our quantitative comparison between pn panel and culture results was somewhat limited by differences in the range of values reported by each method. the pn panel has a broad dynamic range and assigns a bin value of , , , or Ն copies/ml, whereas routine soc culture reports values spanning a narrower range, including , , or Ն cfu/ml. therefore, a direct log bin comparison was possible only for values reported at copies or cfu/ml. values of cfu/ml in culture were considered concordant if reported as "not detected" by the pn panel, and values of Ն cfu/ml in culture were considered concordant if reported as , , or Ն copies/ml by the pn panel. based on these criteria, there was only . % "log bin" quantitative concordance between the pn panel and soc for culture-positive specimens. a % ( / ) concordance was observed for targets reported as Ն cfu/ml in culture ( , , or Ն copies/ml by the pn panel), while log bin concordance was just % to % for targets reported at or cfu/ml, respectively. the relatively poor absolute quantitative correlation between the pn panel (copies per milliliter) and culture (cfu per milliliter) results was noted in the clinical trial data set and is acknowledged as a limitation in the pn panel product labeling ( ) . however, if a categorical-agreement model for treatment based on suggested thresholds of cfu/ml for bal is considered, then the pn panel and culture demonstrate . % ( / ) categorical agreement with no very major errors; i.e., pn panel quantification was never Ͻ copies/ml when culture results were Ն cfu/ml. in addition to absolute quantitation of individual targets, the relative abundance of each organism in a polymicrobial specimen may be of value when therapy is being targeted to the most likely pathogen ( ) . to that end, we examined the agreement between the pn panel and culture for reporting the same organism as predominant (i.e., most abundant) in a clinical specimen. among culture-positive specimens, we observed over % agreement between the pn panel and culture for this comparison. taken together, these data demonstrate excellent categorical and relative abundance agreement between the pn panel and culture methods despite low absolute quantitative correlation (i.e., same log value). when the clinical utility of the pn panel result is being considered, relative abundance and predominance of a given organism are more likely to drive patient management based on current guidelines than the exact quantitative value. however, it will be important to educate clinicians on the expected differences in absolute value observed between culture results in cfu per milliliter and the pn panel results in copies per milliliter to avoid potential confusion when these values are compared and applied to current culture-based guidelines. molecular detection of genetic markers associated with antibiotic resistance, including meca, carbapenemases, and esbls, has been associated with positive outcomes, including reduced time to optimal antibiotic therapy, shorter length of icu stay, and reduced mortality ( , ) . furthermore, many health care systems employ specific contact isolation policies for patients harboring resistant bacteria containing these markers. among specimens that were culture-positive for s. aureus, the pn panel demonstrated a modest . % sensitivity and . % specificity for identification of mrsa based on the detection of meca/mecc and mrej. false-negative results may represent mrsa isolates that contain divergent sequences within the mrej region targeted by the pn panel. alternatively, these cultures may contain a mixture of mrsa and mssa, with mrsa falling below the pn panel limit of detection (lod). specimens containing s. aureus with the mrej sequence but lacking meca (i.e., "meca dropout"), in addition to methicillin-resistant coagulase-negative staphylococcus spp., could result in an apparent false-positive result. each of these scenarios was encountered during the clinical trial, which reported a higher postresolution sensitivity of . % and specificity of . % for detection of mrsa in bal specimens ( ) . importantly, our cohort included just specimens that were culture positive for s. aureus. evaluation of a larger number of mrsa-positive specimens and characterized isolates is needed to provide a more complete assessment of the pn panel for the identification of mrsa. nonetheless, a positive meca/ mrej detection by the pn panel was reported for / ( . %) of culture-negative specimens. identification of mrsa in these additional patients has the potential to ensure appropriate antibiotic therapy in patients whose therapy might have otherwise been de-escalated based on negative cultures. furthermore, while negative culture results likely limit the risk of transmission, detection of mrsa also enables implementation of infection prevention practices in accordance with existing hospital policies. a low incidence of phenotypic carbapenem resistance was observed in this study. only / ( . %) specimens contained isolates that exhibited mics indicating carbapenem resistance, including p. aeruginosa (n ϭ ), acinetobacter baumannii (n ϭ ), and e. cloacae (n ϭ ). the pn panel detected specific carbapenemase genes in the specimens containing carbapenem-resistant e. cloacae or a. baumannii; however, no carbapenemase genes were detected by the pn panel in the specimens containing carbapenem-resistant p. aeruginosa. while we cannot rule out a false-negative pn panel result for carbapenemase detection, carbapenem resistance in p. aeruginosa is most often mediated by mechanisms other than carbapenemases (e.g., efflux pumps or expression of altered outer membrane porins) ( ) . therefore, molecular detection of carbapenemase-encoding genes in non-enterobacteriaceae has a limited negative predictive value. similarly, detection of bla ctx-m demonstrated a % positive predictive value but a reduced negative predictive value for predicting the presence of esblproducing bacteria in bal specimens due to the diversity of enzymes capable of conferring this phenotype. these are important limitations that extend to all molecular diagnostic tests that target specific genetic markers to predict phenotypic susceptibility. therefore, failure to detect a specific gene should not be interpreted as phenotypic susceptibility. conversely, detection of a specific genetic marker carries a high probability of phenotypic resistance to the corresponding antibiotic class. these detections can aid in both rapid antibiotic escalation when indicated as well as implementation of appropriate infection prevention practices for these patients. the role of viral pathogens in hospital-acquired pneumonia (hap) and ventilatorassociated pneumonia (vap) has only recently been appreciated, in part due to the increased availability of multiplex molecular panels to detect these agents. a recent study identified a viral pathogen in . % of patients being evaluated for hap with the risk of a viral infection increasing with duration of icu stay ( ) . interestingly, no seasonal trend in positivity was noted, suggesting that viruses should be routinely considered in this patient population. in our study, the pn panel detected a viral pathogen in approximately % of specimens tested. human rhinovirus/enterovirus was the most frequently detected viral target, but all pn panel viral targets were identified in at least one clinical specimen. of interest, only one-third of specimens had a clinical test order for viral pathogens, including only one-quarter of specimens with a virus detected by the pn panel. the lack of a uniform soc comparator test order and result for the majority of enrolled specimens precludes a thorough comparative assessment of the pn panel performance for detection of viral targets; however, the clinical trial demonstrated Ͼ % positive agreement and Ͼ % negative agreement between the pn panel results and reference pcr and sequence analysis for each target ( ) . importantly, our data demonstrate the infrequency of clinician orders for tests to detect common respiratory viruses in this patient population, which could contribute to underdiagnosis or delayed diagnosis of potentially treatable infections. specifically, only / bal specimens that were reported as positive for influenza a or b virus by the pn panel had a standard-of-care order for a test capable of detecting these pathogens. early recognition of influenza virus infections might have resulted in administration of antiviral therapy that could have shortened the duration and severity of symptoms in these patients ( , ) . while no specific therapy is available for the majority of viral pathogens, . % ( / ) of specimens with a viral detection did not have a bacterial target codetection, supporting the potential contribution of these viruses to respiratory symptoms observed in these patients. in these patients, definitive identification of a viral agent in the absence of a bacterial pathogen could enable an opportunity for antibiotic stewardship. furthermore, patients with underlying comorbidities are at risk of severe infection by human metapneumovirus, parainfluenza viruses, and others ( , ) . recognition of these infections in icu patients enables appropriate infection prevention practices, including droplet isolation to prevent subsequent hospitalacquired infections in a susceptible population. combined, these results support the added value of viral targets as part of multiplex panels designed to aid in diagnosis of lower respiratory tract infections, including hap and vap. a major goal of our study was to examine the potential impact of the pn panel results on antibiotic utilization. for specimens with positive or negative agreement between the pn panel and routine culture, it was assumed that the antibiotic modifications made based on routine culture results would also be made based on the pn panel result. based on review of patient charts, we found that antibiotic adjustments could have been made for over % of patients who submitted respiratory specimens for this study. most commonly, this involved discontinuation or de-escalation of empirical therapy and encompassed approximately % of all antimicrobial modifications and nearly % of patients included in the analysis. combined, this equated to a total of Ͼ , h of antibiotic sparing, or an average of . days/antibiotic. this . -day differential essentially equates to the time difference between availability of the pn panel result and the final culture result. importantly, while it was less common, we did find . % of patients with results indicating that they were receiving ineffective antibiotic therapy for pathogens or resistance markers detected by the pn panel and ultimately reported by routine culture. notably, / of these were detection of influenza a or b virus by the pn panel in patients without a clinician-ordered test capable of detecting these viruses. early recognition of antibiotic mismatches can be potentially lifesaving, as demonstrated by an increased mortality rate observed for patients receiving ineffective or delayed therapy ( , ) . as discussed above, it is difficult to determine the clinical significance of pn panel detections in culture-negative specimens. for this analysis, we chose to be conservative and consider these "false-positive" pn panel results potentially leading to inappropriate or unnecessary antibiotic initiation/escalation. using these criteria, approximately % of patients reviewed would have received antibiotics that were not prescribed based on routine culture results. importantly, the pn panel is an adjunct to early decision-making and does not replace clinical assessment and review of other laboratory values. it is reasonable to assume that a single target present at copies/ml in a patient with minor clinical symptoms or alternative etiology might not result in antibiotic treatment based solely on the pn panel result. however, using conservative criteria assuming that all detections lead to antibiotic prescriptions, the overall data still demonstrate a : ratio of appropriate to inappropriate antibiotic modifications based on pn panel results. of note, antibiotic therapy for % of patients would not have been modified based on the pn panel result; however, early confirmation that these patients were receiving optimal therapy might also have been beneficial in managing care. this study did have limitations. first, it was assumed that all specimens were obtained from patients with clinical symptoms concerning for pneumonia. it is possible that some of the specimens could have been collected as part of surveillance protocols from asymptomatic patients who had recently undergone transplant procedures. second, pn panel results were compared to the routine soc methods and results reported by seven different clinical laboratories, each of which used slightly different specimen processing, plating, and reporting protocols. furthermore, we relied solely on results of clinician-ordered tests, which were absent for approximately % of specimens in the context of viral etiologies. a full clinical and analytical evaluation is reported elsewhere ( ) but does not address how pn panel results compare to soc methods currently used in clinical laboratories. this study was specifically designed to be a pragmatic comparison of pn panel results to soc test results to examine how implementation of the pn panel might impact qualitative positivity and quantitative result reporting. our data raise awareness of specific aspects of the pn panel results, including qualitative and quantitative concordance as well as identification of additional bacterial and viral pathogens that can be expected following implementation of the pn panel, so that laboratory directors and clinicians can begin to discuss utilization and result reporting based on this novel test approach. one of the key findings was the potential for a large impact on antibiotic adjustments, primarily de-escalation and discontinuation, based on the pn panel result. these results were similarly limited by the retrospective design of the study; however, we took a conservative approach, considering adjustments appropriate only when positive or negative agreement was observed between pn panel and soc results. the actual impact of these potential adjustments on quality indicators such as length of stay, -day mortality, and readmission rate could not be assessed based on retrospective analysis and will require prospective randomized controlled trials. still, the potential for early recognition and de-escalation or discon-tinuation of antibiotics in specimens that test negative by pn panel and subsequent routine culture is attractive for meeting antibiotic stewardship goals. notably, we considered only adult inpatients and only bal or mini-bal specimens in this study. therefore, we have not evaluated the potential impact of the pn panel results in pediatric or ambulatory outpatient populations or with other validated specimen types, such as sputum or sputum-like (e.g., endotracheal aspirate) specimens. given the greater acuteness of illness in hospitalized patients, we chose to focus on the benefit of the pn panel in this population. similarly, we chose to focus on bal specimens, because they are frequently of better quality and results have a higher predictive value for lower respiratory tract infection than those obtained with sputum specimens, which may more frequently contain higher bacterial burdens and diversity of insignificant upper airway or oral flora. preliminary data ( , ) did demonstrate a higher percentage of positive specimens among sputum specimens than bal specimens, though clinical data and the impact of these detections on antibiotic stewardship or potential patient outcome were not assessed. therefore, there remains a need for similar studies focusing on these alternative populations and specimen types. in conclusion, we present the first "real-life" assessment of the potential impact of the pn panel results on reporting compared to routine soc culture and clinicianordered viral pcr tests, as well as the potential for antibiotic stewardship based on these results. laboratories considering implementation of the pn panel will have to give careful consideration to reporting and interpretation of results because of the increased sensitivity and differences in absolute quantification between pn panel and soc methods. clinician education will also be an important component of successful implementation of the pn panel to ensure that results are interpreted in the context of other laboratory results. laboratories may choose to report discrete semiquantitative bin values with clinician understanding that these will be higher than current culture-based quantitative results. alternatively, laboratories may choose to report relative abundance of bacterial targets based on pn panel results, assigning each log bin a numeric value from to so that the clinician can quickly assess the predominant pathogen(s) in the specimen. likewise, education focused on the interpretation of results of genotypic resistance, including the positive and negative predictive value of markers, will be important to maximize antibiotic stewardship efforts and ensure appropriate adjustments. specifically, negative results do not preclude phenotypic resistance due to alternative mechanisms. as with other rapid diagnostic tests, maximizing the impact of pn panel results involves the utilization of additional resources, including electronic decision support to ensure appropriate utilization, inclusion of interpretive comments in the lab report to aid interpretation, and inclusion of an active interventional antimicrobial stewardship team ( ) ( ) ( ) . the pn panel will not be used as a replacement for routine culture. however, as an adjunctive test for patients with symptoms of lower respiratory tract infection, the pn panel has the potential to provide rapid identification of bacterial and viral pathogens that can be used to aid in definitive etiologic diagnosis and positively impact efforts to meet infection prevention and antibiotic stewardship objectives. supplemental material is available online only. supplemental file , pdf file, . mb. biofire diagnostics, llc, provided materials and funding to support the clinical evaluation of the pn panel from which the data for this paper were derived. b. w. buchan has received speaker's honoraria from biofire; a. harrington has received speaker's honoraria and research funding and is a member of the biofire scientific advisory board; j. dien bard is a consultant to biofire; c. graue is employed by biofire. global, regional, and national age-sex specific all-cause and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study hospital-acquired pneumonia and ventilator-associated pneumonia: recent advances in epidemiology and management management of adults with 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buzyn, agnès; dreyfus, françois; cariou, alain; freymuth, françois; lebon, pierre title: coronavirus e-related pneumonia in immunocompromised patients date: - - journal: clin infect dis doi: . / sha: doc_id: cord_uid: j lfq o coronaviruses strains e and oc have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. here we report well-documented cases of pneumonia related to coronavirus e, each with a different clinical presentation. diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase—polymerase chain reaction. on the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients. pulmonary complications occur frequently in immunocompromised patients who are treated for hematological malignancies, and they cover a wide range of etiologies, including infections of bacterial, fungal, parasitic, or viral origin; pulmonary edema; diffuse alveolar hemorrhage; drug-or radiation-induced toxicity; graft-versus-host disease; and bronchiolitis obliterans [ ] . in addition, an entity named "idiopathic pneumonia syndrome," defined as diffuse lung injury for which the etiology is not identified, is frequently recognized after hematopoietic stem cell transplantation (hsct). a large number of these cases of pneumonia may be related to unrecognized viral infections, and, in the absence of routine screening or reliable diagnostic procedures, this number is probably an underestimate. coronaviruses are positive-sense, single-stranded rna viruses whose particles are irregularly shaped. the outer envelope carries distinctive club-shaped peplomers, giv-ing a crown-like appearance. two coronavirus strains, oc and e, are known to be involved in human diseases, mainly in the common cold syndrome, but also in pneumonia [ ] [ ] [ ] [ ] . coronaviruses recently became a subject of particular interest because a novel strain was identified as the primary agent associated with the worldwide outbreak of severe acute respiratory syndrome (sars) [ ] . we report well-documented cases of coronavirus-related pneumonia in immunocompromised patients who were treated for hematological malignancies. our diagnostic strategy combined viral culture of huh cells and rt-pcr controlled by hybridization with strain-specific probes. a -year-old white man was admitted to the hospital with fever, weight loss, and continuous nonproductive cough. three years previously, this patient was treated for stage ii large b cell non-hodgkin lymphoma, according to the french lnh protocol [ ] . because of disease progression, he underwent highdose chemotherapy intensification supported by autologous hsct. long-term, complete remission was achieved. relapse occurred years after transplantation and was treated with high-dose sequential salvage therapy combining etoposide, ifosfamide, mitoxantrone, and cytarabine. ten days after the second cycle of chemotherapy, disseminated cutaneous vesicles appeared, suggesting varicella zoster virus (vzv)-associated eruptions. no respiratory symptoms or abnormal chest radiograph findings were present. the patient was successfully treated with intravenous acyclovir ( mg/kg every h for days). after completion of acyclovir treatment, the patient developed a febrile nonproductive cough without dyspnea. a chest radiograph showed bilateral interstitial syndrome, and this was confirmed by a ct scan, which showed disseminated micronodular opacities. arterial blood gas values while breathing in room air were normal. other laboratory findings were as follows: wbc count, cells/mm , with polymorphonuclear cells and lymphocytes; platelet count, , platelets/mm . liver enzyme levels were normal. sputum samples were negative for legionella species when subjected to direct immunofluorescence, and bacterial culture of the sputum samples showed no growth. results of tests of serum samples for aspergillus galactomannan antigen were also negative. following failure of empirical antibiotic treatment (piperacillin and ciprofloxacin), fiber-optic bronchoalveolar lavage (bal) was performed in the middle lobe. cytological analysis of specimens of the bal fluid showed nucleated cells/mm , with % polymorphonuclear cells, % macrophages, and % lymphocytes (identified as t-cell lymphocytes by flow cytometry analysis [ % cd + cells and % cd + cells]). the findings of extensive microbiological direct examinations and cultures remained negative for bacteria; mycobacteria; fungi; and parasites, such as pneumocystis carinii. the results of immunofluorescent assays for multiple respiratory viruses (respiratory syncitial virus [rsv]; parainfluenza viruses i, ii, and iii; adenovirus; and influenzae viruses a and b) and herpes viruses (herpes simplex virus [hsv], vzv), and immunoperoxydase staining for cytomegalovirus (cmv) were negative in specimens of bal fluid. the result of a pcr test performed on specimens of bal fluid with vzv-specific primers was also negative [ ] . viral cultures showed no growth in human amniotic cells or in mrc and vero cell lines. in contrast, a cytopathic effect was observed in the human hepatoma huh cell line cultured with specimens of the bal fluid, which was unrecognized with influenza and parainfluenza viruses, rsv, adenovirus, measles, enterovirus, or vzv-specific antibodies. electron microscopy findings identified corona-like particles in the culture supernatant medium ( figure ). an rt-pcr test performed with infected and noninfected huh cells and using strain-specific primers located in the m gene of human coronaviruses confirmed the diagnosis and typed the virus as e strain, but not as oc strain (figure ). rt-pcr products were also characterized as sequences of e virus by hybridization with a specific probe in a dna enzyme immunoassay [ ] . the presence of coronavirus rna in bal fluid specimens was confirmed by amplification of e sequences, whereas the results of rt-pcr performed with oligonucleotide primers derived from the m gene sequence of the oc strain remained negative. the results of cytopathic effect seroneutralization testing showed a low initial antibody response but a significant difference between the antibody response of preinfection serum samples (titer, ! ) and that of postinfection serum samples (titer, ). all serological studies for respiratory viruses (adenovirus, myxovirus, paramyxovirus, and rsv), cmv, vzv, epstein-barr virus, hsv, and mycoplasma species revealed no significant increase in serum sample antibody titer, and the results of serological testing for chlamydiae bacteria and aspergillus remained negative. the outcome was spontaneously favorable, and the fever disappeared in days without modification of the antibiotic regimen, which was consistent with an improvement of the chest radiograph findings. case study . a -year-old female patient was admitted to the intensive care unit (icu) for acute respiratory failure days after allogeneic bone marrow transplantation. she had been treated with mechlorethamine, vincristine, procarbazine, and prednisone (mopp) chemotherapy combination years previously for hodgkin lymphoma with cervical and mediastinal lymphadenopathies. a first relapse with pulmonary and gastric involvement was treated with high-dose chemotherapy intensification (with melphalan, misulban, and cytarabine) supported by autologous hsct. because of a second relapse with pulmonary and gastric involvement, the patient underwent allogeneic bone marrow transplantation with a conditioning regimen combining high-dose cyclophosphamide and fludarabine. after initiation of the conditioning regimen, she presented with bilateral knee arthritis. samples of the joint fluid tested positive for aspergillus fumigatus, and the patient was treated with amphotericin b ( mg/kg per day). four days after transplantation, rapidly progressive respiratory failure developed, leading to hospitalization in the icu. at admission, physical examination revealed acute respiratory distress with tachypnea ( breaths/min), bilateral crackles, and oxygen saturation of % while breathing with a highconcentration oxygen mask, and shock (heart rate, beats/ min; blood pressure, / mm hg) with disseminated mottling. arterial blood gases obtained while breathing oxygen (with a high-concentration oxygen mask at a rate of l/min) were as follows: ph, . ; partial pressure of carbon dioxide, . kpa; partial pressure of oxygen, . kpa; bicarbonates, . mmol/l. laboratory findings revealed pancytopenia (wbc count, cells/mm ; hemoglobin, . g/dl; platelet count, , platelets/mm ), acute renal failure (creatinine level, mmol/l), and inflammatory syndrome (c-reactive protein, mg/l; fibrinogen, . g/l). a chest radiograph revealed disseminated alveolar and interstitial opacities. the patient's condition deteriorated rapidly, requiring endotracheal intubation and mechanical ventilation. results of a test performed on a tracheobronchial secretion sample were positive for a. fumigatus, leading to treatment with a combination therapy of voriconazole and caspofungin. after an initial improvement in clinical characteristics and radiological findings and bone marrow recovery, the patient's respiratory condition worsened, and bilateral alveolar opacities appeared on a chest radiograph. the results of blood tests for aspergillus galactomannan and cmv pp antigens remained negative. specimens obtained by bal performed in the lingular region were hemorrhagic, and the findings of a quantitative cytological examination were noncontributive, because of numerous lysed cells. the findings of extensive research to detect the presence of microorganisms (bacteria, mycobacteria, fungi, parasites, cmv, and rsv) were negative. despite aggressive supportive care with mechanical ventilation, fluid resuscitation, and high-dose norepinephrine infusion, refractory hypoxia rapidly led to fatal multiorgan failure. no autopsy was performed. cultures of bal fluid specimens remained negative for bacteria, mycobacteria, and fungi. the same procedures for viral culture and rt-pcr were applied as in case study , described above. the results of inoculation tests performed with huh cells were also positive, revealing corona-like particles that were subsequently identified as coronavirus e by rt-pcr performed on both culture supernatant and bal fluid specimens. in both cases, major facts led to the diagnosis of coronavirus e-associated pneumonia: first, the exclusion of alternative infectious etiologies, and, second, the identification of coronavirus e in bal fluid samples by a combination of culture, electron microscopy, and rt-pcr. in the case of the first patient, the main differential diagnosis was vzv-associated pneumonia, which is the most frequent complication of varicella in adults and is usually concomitant to cutaneous vesicles. however, respiratory symptoms only appeared after completion of antiviral treatment and improvement of skin eruptions, and both viral culture and pcr for vzv performed on bal fluid specimens were negative. coronavirus seroconversion retrospectively confirmed the diagnosis. the second patient presented with severe acute respiratory failure that led to fatal multiorgan failure, despite maximal life support. ventilator-associated pneumonia is a frequent complication in patients receiving mechanical ventilation, and it is associated with a high mortality rate, especially among bone marrow recipients. bacterial etiology is largely predominant in ventilator-associated pneumonia, but cmv has been identified as a possible cause in adult patients, and outbreaks of coronavirus-and adenovirus-related pneumonia in pediatric icus have been described [ ] [ ] [ ] . in the case we describe, though negative results of bacteriological cultures cannot exclude a bacterial origin, a secondary identification of coronavirus in bal fluid specimens provided a high probability that a diagnosis of viral origin was accurate. various methods to detect coronaviruses exist, but they lack sensitivity or specificity when used for routine screening of respiratory samples. these methods include identification of corona-like particles, immunofluorescent assays with monoclonal antibodies, and rna hybridization in fluid specimens from nasal washing or bal [ ] [ ] [ ] . detection of seroconversion, based on elisa findings, may be useful, but it does not allow rapid virus-identification and only provides a retrospective diagnosis [ ] . we identified coronavirus in both patients because the bal specimens were cultured in the human hepatoma huh cell line, which expresses a specific receptor for coronaviruses [ ] . recent advances in virological detection methods, such as molecular amplification techniques, offer promising perspectives for detection of coronaviruses [ ] . indeed, improved sensitivity and specificity have been obtained with molecular detection methods combining rt-pcr with strain-specific primers derived from the m protein gene, followed by molecular hybridization with specific probes recognizing either coronavirus e or oc [ ] . inversely, the high sensitivity of amplification methods may lead to false-positive results caused by contamination of bal fluid specimens with pharyngeal viruses. therefore, the combination of viral culture and rt-pcr of bal fluid samples appears to be an efficient and reliable diagnostic strategy. two coronavirus strains, e and oc , have previously been related to human diseases with various clinical syndromes, ranging from self-resolving common cold to pneumonia [ ] [ ] [ ] [ ] . recently, the worldwide outbreak of sars was linked to a novel coronavirus that had not been previously identified in human beings or animals [ ] . the clinical features of sars combine flu-like symptoms, dry cough, and shortness of breath, associated with pulmonary infiltrates visible by chest radiography. in contrast to the common benign symptoms of coronavirus infections in healthy individuals, a large proportion of patients with sars have severe respiratory failure requiring ventilatory support in the icu. the impact of a respiratory virus on individuals is largely determined by their underlying conditions, and particularly by whether they are experiencing immunosuppression [ , ] . the prevalence of coronavirus pulmonary infections among immunocompromised patients is unknown, and it is probably largely underestimated in the absence of the routine performance of sensitive cell culture, rt-pcr, or electron microscopy on bal fluid specimens. however, rt-pcr results were negative for coronaviruses in the bal samples of hsct recipients with acute pulmonary infiltrates [ ] . thus, only case of coronavirus-associated pneumonia was previously described in an immunocompromised patient following autologous bone marrow transplantation, with the diagnosis based on the presence of viral particles in bal fluid specimens [ ] . the identification of coronavirus in high-risk immunocompromised patients may lead to early adoption of a specific therapeutic strategy, but, in the absence of proof of the efficacy of antiviral drugs, the treatment remains only symptomatic. in these circumstances, different types of ifns that display antiviral properties against coronaviruses may be evaluated [ , ] . pulmonary complications of bone marrow transplantation coronavirus infections in working adults. eight-year study with e and oc coronavirus infections in military recruits. three-year study with coronavirus strains oc and e viruses and bacteria in the etiology of the common cold an 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key: cord- -cl kezes authors: poletti, venerino; casoni, gianluca title: procedure diagnostiche invasive nelle malattie infiltrative diffuse del polmone date: journal: pneumologia interventistica doi: . / - - - - _ sha: doc_id: cord_uid: cl kezes le malattie polmonari che, già all’esordio clinico e/o nel loro decorso, coinvolgono più di un lobo e caratterizzate dall’accumulo od infiltrazione nel lobulo polmonare secondario di sostanze o cellule non normalmente presenti in tale sede o presenti, comunque, in quantità anomala, possono essere definite con il termine di pneumopatie infiltrative diffuse (pid) [ ]. le malattie polmonari che, già all'esordio clinico e/o nel loro decorso, coinvolgono più di un lobo e caratterizzate dall'accumulo od infiltrazione nel lobulo polmonare secondario di sostanze o cellule non normalmente presenti in tale sede o presenti, comunque, in quantità anomala, possono essere definite con il termine di pneumopatie infiltrative diffuse (pid) [ ] . il lobulo polmonare secondario, descritto inizialmente da miller nel , è quella struttura parenchimale polmonare che, nelle porzioni più periferiche e sottopleuriche è circondata da setti connettivali completi; esso è comunque composto da tre-cinque acini (unità parenchimale polmonare costituita dal bronchiolo respiratorio e dagli spazi aerei ad esso distali) ed è riconoscibile in tomografia computerizzata ad alta risoluzione (hrct). nelle porzioni centrali del lobulo secondario, decorrono i bronchioli terminali con le associate arterie polmonari, entrambi avvolti in un manicotto connettivale. alla periferia, nei setti connettivali interlobulari, decorrono le vene polmonari. i dotti alveolari, i sacchi alveolari e gli alveoli sono interposti fra queste due aree. i linfatici sono presenti solo nei manicotti connettivali centrolobulari e nei setti interlobulari. la diagnosi delle malattie diffuse del polmone è un processo "a tappe" e le procedure invasive sono prese in considerazione solo quando le altre tecniche non risultino conclusive ed i dati morfologici e/o immunofenotipici ottenibili dalla analisi di tessuto, elementi cellulari o liquido alveolare, sia ritenuta necessaria per una diagnosi definitiva o per le decisioni terapeutiche [ ] . i punti chiave nella discussione del ruolo di tali procedure nel complessivo assetto clinico delle malattie diffuse del polmone sono quindi: . quando una procedura invasiva sia utile o necessaria; . quali procedure invasive risultino essere le più appropriate (rendimento diagnostico atteso, specificità delle informazioni ottenibili, impatto sulle decisioni terapeutiche) ed, eventualmente; . in quale ordine debbano essere effettuate. questi punti sono ancora controversi soprattutto perché il vero impatto dei presidi diagnostici invasivi, stimato prendendo in considerazione la probabilità diagnostica pre-test, non è stato ancora formalmente analizzato. una comprensione delle correlazioni tra anormalità radiologiche e caratteristiche patologiche è stata ottenuta con l'introduzione della tomografia computerizzata ad alta definizione (hrct). procedure diagnostiche invasive nelle malattie infiltrative diffuse del polmone neycombing) si riscontra nelle forme di fibrosi avanzate (fibrosi polmonare idiopatica, collagenopatie evolute). un pattern cistico a distribuzione random può chiamare in causa l'enfisema centrolobulare (cisti senza parete) o l'istiocitosi x (cisti a pareti spesse, con risparmio dei seni costo-frenici) o la linfangioleiomiomatosi (cisti a pareti sottili diffuse senza risparmio dei seni costo-frenici). aree di iperdiafania a chiazze, spesso a distribuzione lobulare, nel cui contesto i vasi sono ridotti di numero e di calibro (oligoemia) si riscontrano nella bronchioliti costrittive o nelle patologie con ostruzione/obliterazione dei piccoli vasi arteriosi polmonari. la hrct dinamica (con scansioni ottenute anche in fase espiratoria) permette in genere di osservare nella patologia delle piccole vie aeree un air-trapping espiratorio, fenomeno questo meno frequentemente osservabile nelle aree di oligoemia a mosaico secondarie a patologia primitivamente vascolare. il potenziale diagnostico differenziale fornito dalla hrct si è dimostrato utile nel predire l'accuratezza diagnostica delle procedure invasive [ ] [ ] [ ] . dopo l'avvento dell'hrct nella pratica clinica il punto di vista secondo cui la valutazione istologica rappresenti il "gold standard" diagnostico è sempre più discusso [ ] [ ] [ ] . infine il repertorio di analisi immunologiche e molecolari che possono essere effettuate su cellule e tessuto è stato ampliato; questo può migliorare significativamente l'accuratezza diagnostica su campioni di tessuto ottenuti con procedure minimamente invasive. il ruolo e il tempo delle diverse procedure invasive nel work-up diagnostico delle malattie diffuse del polmone possono, perciò, essere valutati prendendo in considerazione le correlazioni tra le caratteristiche istologiche e le tecniche di imaging (in particolare l'hrct) ora utilizzate routinariamente. posto che la malattia diffusa del polmone comprende tutti quei disordini che comportano infiltrazione o accumulo di liquido, cellule o matrice extracellulare nelle strutture del lobulo polmonare secondario e che la diagnosi differenziale è vasta e spesso scoraggiante, il primo passo è differenziare tra pazienti immunocompromessi e immunocompetenti [ , ] . l'approccio diagnostico alla malattia respiratoria nel primo gruppo di pazienti rimane una sfida per almeno tre ragioni: ) la acuzie con cui spesso queste malattie insorgono, ) il sempre più frequente riscontro di malattie diffuse del polmone in questi pazienti, e ) il fatto che le nuove procedure di laboratorio fanno aumentare la sensibilità a scapito della specificità. nei pazienti immunocompetenti le procedure diagnostiche non devono essere completate in tempi così brevi e i nuovi test microbiologici di laboratorio hanno solo un ruolo secondario. infatti in questo gruppo di pazienti solo alcune malattie infiltrative diffuse polmonari possono presentarsi acutamente (polmoniti da ipersensibilità, tossicità polmonare da farmaci, polmoniti correlate a esposizioni tossiche -malattia del "silo filler"-, polmonite eosinofila acuta, polmonite acuta interstiziale, e polmonite in via di organizzazione criptogenetica, neoplasie disseminate a primitivo esordio polmonare, malattie rare quali la iperplasia micronodulare dei pneumoniti di secondo ordine) [ ] e le forme infettive (ad esclusione delle micobatteriosi) sono veramente rare. i pazienti immunocompromessi che sviluppano malattia diffusa del polmone hanno di solito un esordio acuto con tosse, dispnea, febbre e spesso ipossiemia rapidamente progressiva [ , , ] . in queste circostanze può essere difficile distinguere infiltrazioni neoplastiche del polmone da coinvolgimento polmonare di malattie collageno-vascolari o vasculiti, da iperidratazione, insufficienza cardiaca, infezioni opportunistiche o effetti tossici della chemioterapia. radioterapia, graft-versus-host disease (gvhd), rigetto acuto e cronico, e disordini polmonari idiopatici (es. proteinosi alveolare in soggetti neutropenici, danno alveolare diffuso, polmoni-capitolo endoscopia bronchiale diagnostica dell'adulto te in via di organizzazione) possono anche contribuire allo sviluppo di complicanze polmonari in questo scenario clinico. una diagnosi tempestiva sembra anche essere cruciale per una migliore sopravvivenza [ , ] . l'espettorato indotto ha un ruolo solo nei pazienti hiv-positivi per la sua alta resa diagnostica nella polmonite da pneumocystis jirovecii e nella tubercolosi [ , ] . il lavaggio broncoalveolare (bal), soprattutto quando il broncoscopio è guidato dai reperti dell'hrct eseguita poche ore prima, è generalmente di valore nella diagnosi di infezioni opportunistiche, proteinosi alveolari, emorragia alveolare e capillariti, infiltrazione polmonare leucemica (fig. ) o linfomatosa, linfangite carcinomatosa, metastasi polmonari ematogene diffuse, polmoniti da ipersensibilità, polmoniti eosinofile, o danno alveolare diffuso dovuto a radiazioni o farmaci [ ] [ ] [ ] [ ] . aspetti morfologici di per sé diagnostici sono: ammassi di p. jiroveci (fig. ) , presenza di bacilli alcool acidi resistenti (con più precisa tipizzazione con test di biologia molecolare), identificazione con test di immunofluorescenza diretta della legionella pneumophila, presenza di inclusioni virali intracitoplasmatiche o intranucleari (fig. ) (con più precisa tipizzazione utilizzando test di immunocitochimica), strongyloides stercoralis o altri agenti parassitari, di cellule neoplastiche, emosiderina intracitoplasmatica o un recupero di liquido vieppiù emorragico, presenza di eosinofili nella polmonite eosinofila, di macrovacuoli intracitoplasmatici e gocce di olio red-positive intra-ed extra-cellulari nella polmonite lipoidea, di materiale amorfo extracellulare pas-positivo e alcian-negativo nelle lipoproteinosi alveolari, pneumociti di tipo ii reattivi raggruppati in pseudopapille attorno a materiale amorfo extra-cellulare nel danno alveolare diffuso. il ritrovamento di batteri o virus o funghi dopo aver utilizzato le più sofisticate indagini colturali o di biologia molecolare senza corrispettivo morfologico non è di per sé diagnostico di polmonite. in questi casi la distinzione tra polmonite e colonizzazione è basata sugli aspetti radiologici e sul contesto clinico [ , ] . caratteristiche non specifiche nel bal possono essere di aiuto alla diagnosi insieme a un'estesa indagine clinica e radiologica [ , [ ] [ ] [ ] . nella tossicità da farmaci o radiazioni accanto ai cambiamenti iperplastici/displastici dei pneumociti di tipo ii e le indagini microbiologiche negative, è stata descritta ogni tipo di alveolite nel liquido del bal. comunque, linfociti cd + morfologicamente attivati che indicano una reazione di ipersensibilità sono il reperto più frequente e suggestivo. la polmonite interstiziale cellulare come manifestazione clinico-patologica di gvhd nel polmone può essere sospettata sulla base di indagini microbiologiche negative e linfocitosi cd + nel liquido di bal [ ] [ ] [ ] . l'applicazione di sedazione profonda e l'utilizzo di maschera laringea sembra essere una sicura ed efficace alternativa all'intubazione per effettuare fibrobroncoscopia con bal in pazienti (adulti e bambini) con sospetta polmonite e severa ipossiemia [ ] [ ] [ ] . il brushing protetto e il broncolavaggio hanno una accuratezza diagnostica e specificità inferiori poiché non permettono di correlare i dati microbiologici con il profilo citologico rilevato nelle vie aeree respiratorie e il brushing è associato con una più alta incidenza di effetti collaterali, in particolare emorragie e pneumotorace [ ] . i pattern hrct che si sono mostrati predittivi di una più alta accuratezza diagnostica della procedura del bal sono [ , , , ] : opacità alveolari e/o di ground-glass (pattern non specifici osservati più frequentemente nelle infezioni, tossicità farmaco-indotta, polmonite organizzante, emorragia alveolare e danno alveolare diffuso), pattern ad "albero in fiore" (più tipicamente osservato nelle bronchioliti e peribronchioliti da causa in- endoscopia bronchiale diagnostica dell'adulto fettiva, es. tubercolosi, polmoniti lobulari), pattern nodulare o reticolonodulare con una distribuzione perilinfatica (tipicamente osservati nelle infiltrazioni linfomatose e nelle linfangiti carcinomatose), e noduli escavati (di solito di natura infettiva) [ , ] . una diagnosi conclusiva non è ottenuta nella minoranza dei casi. in questi pazienti la biopsia polmonare transbronchiale o anche la biopsia a cielo aperto devono essere considerate. la biopsia polmonare transbronchiale (tbb) è una procedura in cui lo pneumotorace e l'emorragia bronchiale rappresentano gli effetti collaterali più frequenti e pericolosi (osservati comunque in < % dei casi) [ ] . nei casi in cui una tbb sia ritenuta necessaria e praticabile (assenza di significativa coagulopatia, piastrine > , pazienti senza precedente intervento di pneumonectomia, etc.), le procedure broncoscopiche possono essere effettuate in anestesia generale con broncoscopio rigido per un miglior controllo della ventilazione e del sanguinamento [ ] . in questo modo le pinze bioptiche non sono introdotte attraverso il canale operativo del fibrobroncoscopio e perciò, i campioni, frequentemente più larghi delle valve della pinza, non vengono lacerati. in questo modo si possono utilizzare anche pinze di più grandi dimensioni (nella nostra esperienza pinze da duodenoscopio modificate con shaft di circa , mm). il controllo della eventuale emorragia può essere completato con l'aiuto di un aspiratore rigido da mm e l'utilizzo di palloncini di fogarty (tabella , fig. ). bal e tbb in combinazione possono anche essere eseguiti in maniera sicura nei pazienti ventilati meccanicamente [ ] . modalità di esecuzione della biopsia tranbronchiale con broncoscopio rigido . paziente in sedazione profonda utilizzando propofol con o senza remifentanest . intubazione con broncoscopio rigido con diametro interno superiore a cm . introduzione dell'ottica rigida,dell'aspiratore rigido da mm di diametro e di palloncino di fogarty posizionandolo poco sopra il bronco o i bronchi segmentari prescelti come distretto da sottoporre a biopsia . introduzione della pinza e effettuzione delle biopsie nel distretto prescelto sotto controllo fluoroscopico.la pinza dopo essere stata chiusa viene recuperata senza che passi all'interno di canali operativi. . controllo della eventuale emorragia con l'aspiratore e con il palloncino gonfiato alterazioni patologiche non specifiche sono comuni nei campioni ottenuti da tbb in questi pazienti, ma se interpretati nel contesto di uno specifico assetto clinico e dei pattern hrct, possono contribuire alla definizione di una diagnosi specifica (in particolare i quadri di polmonite intersitiziale cellulata associata o meno alla presenza di granulomi con eventuale presenza nell'in-procedure diagnostiche invasive nelle malattie infiltrative diffuse del polmone filtrato infiammatorio di eosinofili, di organizzazione endoalveolare, di danno alveolare diffuso osservabili in diversi contesti: tossicità polmonare da farmaci (fig. ), soggetti con dermatomiosite/polimiosite o altre connettiviti o in pazienti trapiantati) [ , ] . la tbb è a volte di aiuto nel dirimere fra colonizzazione e vera polmonite in casi in cui nel bal siano evidenziati microrganismi patogeni facoltativi (fig. ). il rendimento diagnostico della tbb in questo contesto clinico è comunque molto discusso. katzenstein ed askin [ ] in una serie di biopsie ottenute in pazienti immunodepressi osservarono alterazioni morfologiche nel % dei campioni ma soltanto nel % dei prelievi fu generata dagli autori una diagnosi anatomopatologica autonoma.anche le infezioni possono avere alterazioni non specifiche come unici reperti morfologici. colorazioni speciali sono quindi indispensabili quando campioni da tbb sono valutati nei pazienti immunodepressi [ ] . speciali colorazioni sono anche utili nella classificazione di tumori epiteliali e indispensabili per una miglior identificazione di infiltrati linfoidi. ora stabilire il valore di reperti morfologici non specifici è un problema non tecnico ma concettuale: molte malattie possono avere ed hanno un background morfologico non specifico; tuttavia la associazione fra clinica, imaging e morfologia è sicuramente in grado di fornire dati utili per una approccio non solo diagnostico, ma anche terapeutico; questo è vero soprattutto nei casi di danno polmonare da farmaci, nella diagnosi di lesioni interstiziali gvhd correlate (fig. ) , nella diagnosi di rigetto polmonare. inoltre i prelievi chirurgici non riescono a trasformare un pattern istopatologico, di per sé non specifico, in pattern specifico; in tale maniera si diminuisce solo la probabilità di non prelevare aree contenenti lesioni altrimenti specifiche. questa probabilità elevata circa anni fa oggi è comunque molto ridotta se si prendono in considerazione i dati ottenibili con hrct. [ , ] . la biopsia polmonare chirurgica (di solito per via toracoscopia video-assistita o vats) è effettuata nella residua minoranza dei casi. white et al. [ ] hanno riportato la loro esperienza nei pazienti ematologici. una diagnosi specifica è stata trovata in ( %) biopsie. il fattore più predittivo per giungere ad una diagnosi specifica era la presenza di un'alterazione radiologica focale piuttosto che diffusa. le diagnosi polmonari specifiche finali erano malattie infiammatorie nel % dei casi, infezioni nel %, e neoplasie nel %. il pattern istologico di polmonite in via di organizzazione era l'aspetto morfologico più frequentemente osservato e funghi e batteri erano i più frequenti patogeni infettivi. i pazienti neutropenici o quelli sottoposti a ventilazione meccanica avevano una più bassa probabilità di giungere ad una diagnosi specifica. l'aver ricevuto chemioterapia pneumotossica nei mesi precedenti la biopsia era associato al ritrovamento di danno polmonare non specifico [ ] . modificazioni terapeutiche sono state apportate nel % dei pazienti dopo i risultati della biopsia, ma nel % di quelli con una dia- gnosi specifica, e la sopravvivenza a e giorni era aumentata in quelli con una diagnosi polmonare specifica piuttosto che non specifica [ ] . complicanze sono comparse nel % dei pazienti, compresi pazienti che hanno avuto necessità di ventilazione meccanica dopo la procedura; un decesso è stato associato alla biopsia. il rischio era aumentato in coloro con meno di piastrine. le complicanze sono simili con la vats confrontata con la toracotomia. alcuni autori hanno obiettato che la perdita di impatto delle tecniche diagnostiche invasive sulla sopravvivenza era la motivazione principale per non effettuarle in pazienti immunocompromessi [ ] . tuttavia, rano et al. [ ] hanno trovato una significativa diminuzione della mortalità in quei pazienti con un'eziologia infettiva in cui una diagnosi precoce ( giorni dalla comparsa di infiltrati polmonari) ha condizionato un cambiamento terapeutico ( %) in confronto con quelli in cui la diagnosi era ottenuta dopo giorni ( %). lo stesso gruppo ad un'analisi multivariata nei pazienti immunocompromessi con infiltrati polmonari ha evidenziato che la necessità di ventilazione meccanica, un punteggio apache ii (acute physiology and chronic health evaluation) > e ancora un ritardo > giorni nello stabilire una diagnosi specifica, sono le variabili associate con la mortalità. il valore aggiunto della tbb nei pazienti sottoposti a bal rimane controverso. in uno studio retrospettivo su pazienti immunocompromessi, la tbb è risultata più sensibile del bal ( % vs % nella malattia da hiv, % vs % nelle neoplasie ematologiche, % vs % nei riceventi trapianto di rene) e con poche e gravi complicanze [ ] . nei pazienti con infezione da hiv è stato affermato che un risultato bal negativo suggerirebbe una ripetizione del bal con tbb nel sito con maggiori alterazioni [ ] . in uno studio retrospettivo su pazienti ventilati meccanicamente, la tbb era diagnostica nel % dei casi e ha portato ad un cambiamento della gestione nel % dei pazienti "medici" e nel % dei pazienti con trapianto polmonare [ ] . la frequenza di pneumotorace era più alta ( %) di quella generalmente riportata nei soggetti non ventilati (< %), ma non vi erano serie complicanze [ ] . a successive biopsie a cielo aperto o esami autoptici vi era una concordanza dell' % con i reperti da tbb. gli autori affermano [ ] che la tbb è sicura nei pazienti ventilati meccanicamente con infiltrati polmonari non diagnosticati e può evitare il ricorso alla biopsia polmonare chirurgica. la raccomandazione è quindi di utilizzare procedure semplici non invasive come primo step nella diagnosi degli infiltrati polmonari nei pazienti immunocompromessi. queste procedure non invasive devono comprendere antigenemia ematica per specifici agenti infettivi, colture ematiche, dell'escreato e del liquido nasofaringeo così come dell'aspirato tracheobronchiale nei pazienti con ventilazione meccanica. l'utilizzo di tecniche non invasive conduce alla diagnosi nel % dei casi e costituisce una buona alternativa in quei pazienti con controindicazioni ad un'esplorazione broncoscopica. per l'importanza di ottenere una diagnosi con un minimo ritardo, il bal dovrebbe essere sempre eseguito quando possibile poiché tale procedura ha un'alta probabilità diagnostica sia per eziologie infettive che non infettive. l'uso del brushing protetto può essere evitato in quanto non contribuisce ad aumentare la probabilità diagnostica del bal. infine, stabilito che non vi sono controindicazioni, pazienti selezionati devono essere sottoposti ad agoaspirato o biopsie transbronchiali come step prima delle procedure chirurgiche. nei pazienti immunocompetenti lo spettro clinico delle malattie diffuse del polmone è molto vasto. inoltre, nelle ultime due decadi, l'hrct utilizzata insieme alla clinica e altre modalità investigative non invasive ha aumentato l'accuratezza della diagnosi per alcune malattie senza necessità di biopsia chirurgica (la maggioranza dei casi di fibrosi polmonare idiopatica, polmoniti capitolo endoscopia bronchiale diagnostica dell'adulto da ipersensibilità, ipf, istiocitosi a cellule di langherans, linfangioleiomiomatosi, silicosi) e i pattern hrct con cui la malattia si presenta all'esordio, si sono dimostrati molto utili nella scelta delle procedure diagnostiche invasive [ , [ ] [ ] [ ] [ ] ]. l'espettorato indotto ha un ruolo subordinato, essendo diagnostico nei casi di polmonite lipoidea, polmonite eosinofila e, solo in mani esperte e in centri di ricerca, nella sarcoidosi quando il rapporto cd /cd è significativamente alto [ , ] . il bal è attualmente considerato una pietra miliare nel work-up diagnostico dei soggetti immunocompetenti che hanno una malattia diffusa del polmone. in tale contesto ci sono alcune malattie polmonari in cui il bal può fornire specifici reperti che, se presenti, evitano il ricorso alla biopsia: proteinosi alveolare, esposizione a polveri, malattie eosinofile polmonari, emorragia alveolare, danno alveolare diffuso qualunque sia il contesto clinico in cui si sviluppi (polmonite acuta interstiziale, esacerbazione acuta di ipf, ards), istiocitosi a cellule di langherans, linfoma a cellule b tipo malt, linfangite carcinomatosa, carcinoma bronchioloalveolare (fig. ) , polmonite lipoidea, e disturbi più rari, quali le malattie metaboliche congenite (malattia di gaucher, malattia di hermansky-pudlak) [ , [ ] [ ] [ ] [ ] [ ] . È rilevante che molti di questi disordini siano caratterizzati da un punto di vista patologico dall'accumulo di cellule diagnostiche o materiale extracellulare negli spazi alveolari (il cosiddetto gruppo delle malattie da riempimento alveolare) e da un punto di vista radiologico da opacità alveolari o aree a vetro smerigliato all'hrct. la probabilità diagnostica è molto alta (circa % nella proteinosi alveolare e nelle eosinofilie polmonari) eccetto per l'istiocitosi a cellule di langherans in cui più del % delle cellule cd a+ sono ritrovate in meno del % dei casi nella nostra esperienza (e di solito nei casi con tipiche lesioni nodulari e cistiche all'hrct). in un numero maggiore di malattie diffuse del polmone, i reperti del bal non sono specifici, ma possono essere utilizzati come supporto alla diagnosi. in queste condizioni l'analisi del bal può essere la chiave per la diagnosi, insieme ad un'accurata valutazione clinico/radiologica, rendendo una biopsia non necessaria [ ] . comunque, se a dispetto di queste accurate indagini la diagnosi rimane incerta, una biopsia deve essere considerata come ulteriore passo diagnostico. la tbb deve essere considerata come prima scelta. ad andersen et al. [ ] è attribuita la prima descrizione di tbb a seguito della loro osservazione che i campioni di biopsie bronchiali delle piccole vie aeree raccolti in corso di broncoscopia rigida spesso contenevano parenchima polmonare. le pinze da tbb raggiungono il tessuto polmonare attraverso le vie bronchiali e perciò i campioni così ottenuti provengono dalle regioni centrolobulari [ ] . i disordini che fig. a,b . donna di anni con dispnea da sforzo.(a) la tac documenta una ampia area di consolidazione nel lobo inferiore del polmone destro. all'interno sono riconoscibili i vasi polmonari impregnati con mezzo di contrasto (angiogram sign).(b) il bal evidenzia la presenza di papille composte da cellule epiteliali, con nucleolo e disposte su diversi piani (diagnostiche di adenocarcinoma papillifero/carcinoma bronchioloalveolare,diff quick) b a procedure diagnostiche invasive nelle malattie infiltrative diffuse del polmone sono centrati attorno ai bronchioli terminali e respiratori (bronchioliti respiratorie, rb, tubercolosi, polmoniti infettive lobulari, bronchioliti cellulari) o che coinvolgono significativamente queste strutture (polmonite in via di organizzazione) o sono distribuiti lungo le vie linfatiche (sarcoidosi, linfangite carcinomatosa) possono essere facilmente campionate con le pinze. nei disordini con distribuzione linfatica e perilinfatica le biopsie bronchiali possono anche contribuire ad aumentare l'accuratezza diagnostica [ , ] . la polmonite da ipersensibilità nel suo stadio subacuto è istologicamente definita dalla presenza di infiltrato linfocitico peribronchiolare (bronchiolite cellulare), piccoli granulomi mal definiti, e polmonite organizzante [ ] . campioni numerosi e di dimensione superiori ai - millimetri da tbb possono documentare la presenza di questa triade e, perciò, essere diagnostici. la probabilità diagnostica della tbb nella polmonite da ipersensibilità varia da circa il % a più del %, ma questo vasto range di probabilità diagnostica è forse dovuto ai differenti criteri istologici accettati, al numero di campioni bioptici raccolti [ , ] e alle cause di polmonite (essendo i granulomi più frequentemente riscontrabili nel polmone del contadino). altri pattern istologici monomorfi (polmonite organizzante, polmonite eosinofila, danno alveolare diffuso con o senza eosinofili, emorragia alveolare con o senza capillarite, proteinosi alveolare) [ , ] sono facilmente identificabili in piccoli frammenti di polmone, ma sono più o meno non specifici e sono considerati diagnostici solo in accordo con i dati clinici, radiologici e di laboratorio. le vasculiti polmonari si manifestano con aspetti morfologici variegati: necrosi a carta geografica, background infiammatorio, organizzazione endoalveolare, capillarite, vasculite neutrofilica e a cellule giganti o con prevalenza di eosinofili, infiltrati eosinofili. la biopsia chirurgica è raramente necessaria (specialmente nei casi in cui il laboratorio abbia in precedenza dimostrato la presenza di autoanticorpi anti-neutrofili, anca). il ruolo della biopsia transbronchiale è discusso: casi anedottici di angioite di wegener o di churg strauss con lesioni morfologiche caratteristiche su frammenti bioptici transbronchiali sono rintracciabili in letteratura. anche in questo campo il valore diagnostico e terapeutico di una biopsia dipende dal contesto clinico: la presenza di aspetti morfologici in piccoli prelievi caratteristici ma non completamente esaustivi della forma di vasculite sospettata possono essere ritenuti sufficienti per impostare una terapia impegnativa con steroidi e immunosoppressori. con la tbb è possibile ottenere prelievi con aspetti di per sé diagnostici in: linfangite carcinomatosa, metastasi da neoplasia maligna, granulomatosi a cellule di langerhans, linfangioeliomiomatosi, proteinosi alveolare, processi linfoproliferativi, infezioni (quando oltre agli aspetti morfologici compatibili sia possibile identificare nel tessuto l'agente infettivo), pneumoconiosi (in particolare silicosi) [ ] . indagini immunoistochimiche possono aumentare la specificità diagnostica [ ] . i piccoli campioni ottenuti con tbb possono non essere sufficienti nei casi in cui le caratteristiche morfologiche siano tipiche solo in ampi volumi (polmonite interstiziale usuale, polmonite interstiziale desquamativa, polmonite interstiziale non specifica varietà fibrosante). dal punto di vista morfologico il pattern di polmonite interstiziale nonspecifica (nsip), variante cellulare, è molto caratteristico (conservazione dell'architettura polmonare, ispessimento interstiziale dovuto a infiltrazione cellulare mononucleare, metaplasia cuboidale tipo ii), ma è clinicamente non specifico essendo osservato in casi di malattie collageno-vascolari (miosite infiammatoria in particolare), nei pazienti con tossicità farmaco-correlata, nei danni polmonari da gvhd, e in una minoranza dei casi ha un'origine sconosciuta (nsip idiopatica) [ ] . la biopsia polmonare chirurgica è considerata la via migliore per ottenere tessuto sufficiente a dimostrare tale pattern. comunque, tbb generose possono consentire buoni prelievi per una diagnosi morfologica (fig. ) . questa diagnosi morfologica è accettabile in specifici contesti clinici (malattia polmonare interstiziale correlata a polimiosite-dermatomiosite, tossicità da farmaci), ma può anche essere di valore clinico nei casi ad eziologia ignota [ ] . rb è facil-mente riconoscibile nei campioni tbb (fig. ) e oggigiorno l'utilità della biopsia polmonare chirurgica nella diagnosi di malattia interstiziale polmonare associata a rb è discutibile e probabilmente eticamente non corretta. per la diagnosi di pattern uip (usual interstitial pneumonia) sono al momento necessarie biopsie chirurgiche; tuttavia, del tutto recentemente, berbesku et al. [ ] hanno riportato che una "patchwork fibrosis" associata a focolai fibroblastici e/o honeycomb lung è presente nel % dei soggetti con ipf sottoposti a tbb. il ruolo del numero dei campioni ottenuti e della grandezza delle pinze non è stato valutato in dettaglio nella letteratura. descombes et al. [ ] hanno rivisto i dati clinici e istologici di tbb consecutive effettuate in pazienti immunocompetenti, che presentavano o infiltrati polmonari diffusi cronici, o una lesione periferica polmonare o adenopatie ilari. gli autori hanno dimostrato che esiste una correlazione diretta tra il numero di campioni ottenuti attraverso tbb e la probabilità diagnostica globale (es. % con - frammenti vs % con - frammenti, p< , ). essi raccomandano che debbano essere prelevati cinque o sei campioni, essendo comunque il numero ottimale di campioni da sette a dieci. curley et al. [ ] hanno riportato che prelievi bioptici più grandi contenevano più probabilmente tessuto diagnostico (r= , , fig. . frammento bioptico transbronchiale. l'architettura parenchimale è conservata ed i setti interalveolari sono infiltrati da cellule mononucleate (pattern nsip variante cellulata,ematossilina-eosina) fig. . biopsia polmonare transbronchiale. un bronchiolo respiratorio con la adiacente arteria polmonare (sul lato destro) presenta il lume irregolare e in parte occupato da macrofagi con pigmento brunastro.i macrofagi sono numerosi anche negli spazi aerei centrolobulari. reperti tipici di "bronchiolite respiratoria"(ematossilina-eosina) procedure diagnostiche invasive nelle malattie infiltrative diffuse del polmone p= , ). le pinze a coppa recuperavano frammenti di tessuto più piccoli (p= , ) ed erano meno idonee a ottenere tessuto diagnostico (p= , ). campioni che galleggiavano erano meno probabilmente diagnostici o anormali rispetto a quelli che affondavano (p< , ). loube et al. [ ] hanno confrontato prospetticamente la resa diagnostica delle biopsie transbronchiali usando pinze larghe e piccole. le pinze grandi hanno ottenuto più tessuto di quelle piccole in maniera significativa ( su pazienti, %, vs su pazienti, %, p< , ). inoltre le pinze grandi hanno ottenuto significativamente più tessuto alveolare rispetto a quelle piccole ( su pazienti, %, vs su , %, p< , con pazienti in cui non è stato ottenuto tessuto alveolare). se le pinze più grandi non vengono chiuse attraverso il canale operativo, possono essere evitati artefatti da lacerazione. becker et al. [ ] e casoni et al. [ ] hanno riportato una significativa resa diagnostica e/o una miglior conservazione dei campioni quando le tbb sono eseguite in anestesia generale utilizzando pinze più grandi e senza tirarle fuori dal canale bioptico (fig. ) . una tec- fig. . alla sinistra pinze di maggiori dimensioni in commercio per fibrobroncoscopi con canale operativo di , mm di diametro con frammento bioptico ottenuto.alla destra pinza bioptica di dimensioni maggiori ottenuta modificando quelle in commercio per duodenoscopi e frammento bioptico ottenuto utilizzandola nica simile (la cosiddetta tecnica non-pull-through) è stata descritta da mullins et al. [ ] nei bambini. utilizzando un catetere a suzione di plastica come canale operativo attraverso cui le pinze bioptiche vengono introdotte e un fibrobroncoscopio flessibile ultrasottile ( , mm) per guidare il catetere visivamente all'interno del segmento polmonare desiderato, gli autori hanno ottenuto tessuto adeguato in otto su nove interventi ( %). i prelievi ottenuti da tbb possono essere sezionati serialmente e indagini immunoistochimiche devono essere incrementate: istiocitosi a cellule di langherans, linfangioleiomiomatosi, sarcoidosi, metastasi carcinomatosa o da neoplasie dei tessuti molli e disordini linfoproliferativi sono meglio definiti utilizzando specifici anticorpi monoclonali (espressione immunoistochimica di cd a, langerin, hmb- , cd , ttf- o cdx- ). nel prossimo futuro probabilmente nuovi marker, identificati sulla base di nuove ipotesi patogenetiche, saranno utili nell'identificazioni di lesioni patologiche in piccoli frammenti [ ] . l'agoaspirato transbronchiale flessibile, una procedura diagnostica broncoscopica minimamente invasiva utilizzata in maniera sempre maggiore nella diagnosi e nello staging del tumore del polmone, e recentemente effettuata dopo ispezione endobronchiale ultrasonografica, ha un ruolo nella diagnosi delle malattie diffuse del polmone quando esse coinvolgano i linfonodi mediastinici all'esordio o lungo il corso della malattia (sarcoidosi, micobatteriosi, disordini linfoproliferativi) [ , ] . si possono effettuare biopsie con pinze flessibili nei linfonodi sottocarenali dopo aver perforato la mucosa bronchiale con un ago gauge ottenendo così frammenti utili per la diagnosi (fig. ) . la biopsia polmonare chirurgica può essere effettuata attraverso una minitoracotomia o durante una vats. la seconda opzione è oggi capitolo endoscopia bronchiale diagnostica dell'adulto preferita a causa della maggiore quantità di tessuto ottenuta e per la possibilità di effettuare prelievi bioptici in più di un sito [ , ] . riduce anche il tempo di permanenza del tubo di drenaggio toracico e del ricovero ospedaliero. comunque non è praticabile in presenza di molteplici aderenze pleuriche o di uno spazio pleurico obliterato o in pazienti che non siano in grado di tollerare la ventilazione monopolmonare. sembra che la biopsia polmonare a cielo aperto per la diagnosi di malattia polmonare diffusa non deteriori la funzionalità polmonare se eseguita attraverso una procedura a cielo aperto o minimamente invasiva [ ] . le regole da applicare quando una biopsia chirurgica sia ritenuta necessaria sono: . il sito della biopsia deve essere scelto tenendo conto delle caratteristiche hrct. gaensler e carrington [ ] hanno affermato che la lingula e l'apice del lobo medio devono essere evitate per il fatto che spesso mostrano fibrosi non specifica. tale questione è ancora controversa; due recenti studi che hanno indagato specificatamente questo quesito non hanno trovato alcuna ragione per cui debba essere evitata la biopsia della lingula e del lobo medio [ , ] . oggi i punti su cui occorre insistere sono l'ausilio dell'hrct nella scelta del sito da biopsiare e che gli apici dei lobi devono essere evitati perché possono mostrare alterazioni fibrotiche o infiammatorie non specifiche. due campioni devono essere prelevati da due differenti lobi; uno da un'area di evidente, ma moderata anormalità, e uno da un'area che appare quasi normale. zone con fibrosi grave (pattern honeycombing all'hrct) non dovrebbero essere biopsiate poiché le lesioni trovate non sono utili per la diagnosi differenziale. . i prelievi da biopsia chirurgica dovrebbero essere fissati in insufflazione. . per assicurarsi che il massimo di informazioni diagnostiche sia ottenuto, una parte della biopsia polmonare dovrebbe essere inviato al laboratorio di microbiologia per colture e colorazioni. una biopsia chirurgica è necessaria per una diagnosi certa di ipf/polmonite interstiziale usuale in casi con reperti hrct atipici, di pattern nsip o di polmonite interstiziale desquamativa, in casi di polmonite interstiziale linfocitaria idiopatica, per confermare una diagnosi di bronchiolite costrittiva idiopatica e iperplasia cellulare neuroendocrina, o in rari casi di malattia infiltrativa diffusa del polmone quando altre procedure non abbiano reso possibile una diagnosi definitiva [ ] . la mediastinoscopia è attualmente raramente utilizzata nel work-up diagnostico delle malattie infiltrative diffuse del polmone e una diagnosi di sarcoidosi in stadio i e/o ii solo con mediastinoscopia senza prima avere provato un approccio meno invasivo può essere criticabile. sono richiesti chirurghi esperti per risultati ottimali. la morbilità e la mortalità sono basse, ma non trascurabili. soprattutto nei pazienti con sospetto clinico di fase accelerata di ipf le biopsie chirurgiche andrebbero evitate poiché associate a elevato rischio di mortalità. la esecuzione della biopsia polmonare chirurgica può altresì essere il fattore scatenante una insufficienza respiratoria acuta. kondoh et al. [ ] , in uno studio retrospettivo dal al , identificarono pazienti con polmonite interstiziale idiopatica in cui fu eseguita per la fig. . biopsia con pinze flessibili da prelievo transbronchiale di linfonodo sottocarenale (durante esame condotto in sedazione profonda e intubazione con broncoscopio rigido) procedure diagnostiche invasive nelle malattie infiltrative diffuse del polmone diagnosi una biopsia polmonare chirurgica; cinque di essi svilupparono una esacerbazione acuta della malattia interstiziale (ipf ; nsip ; cop ) nel decorso postoperatorio ( - giorni). due dei tre soggetti con ipf morirono a causa della complicanza. le procedure invasive praticabili nel work-up diagnostico delle malattie infiltrative diffuse del polmone sono numerose. esistono ancora controversie sul ruolo di queste differenti procedure e sul timing del loro utilizzo. comunque, dopo l'introduzione dell'hrct nella pratica clinica, la valutazione dei pattern di imaging, unitamente allo stato immunologico del paziente e al decorso clinico della malattia (acuto vs cronico) sembra cruciale per risolvere il problema sopra menzionato. nella tabella sono riportati i punti che riassumono il ruolo del bal e della biopsia polmonare transbronchiale nella diagnosi delle pneumopatie diffuse infiltrativi. un algoritmo utile per l'approccio pratico ai pazienti con supposta malattia infiltrativa diffusa del polmone è rappresentato nella figura [ ] . nuove prospettive sono ipotizzabili in base a recenti studi genetici e ai nuovi concetti patogenetici [ ] [ ] [ ] . tabella . i punti chiave delle procedure broncoscopiche nelle pneumopatie diffuse infiltrative • il lavaggio broncoalveolare (bal) deve essere eseguito con almeno ml di soluzione fisiologica;un recupero superiore al % del liquido istillato è ritenuto rappresentare la cellularità del distretto parenchimale polmonare. • recuperi inferiori permettono solo una diagnosi attendibile quando si evidenzino cellule neoplastiche o microrganismi sicuramente patogeni. • la metodica può essere eseguita in soggetti con grave ipossia,ventilati meccanicamente e piastrinopenici (anche con valori di piastrine< ).rischi relativi di complicanze sono presenti in soggetti con aritmie instabili sia dal punto di vista emodinamico che elettrofisiologico,nei pazienti con insufficienza cardiaca sistolica o diastolica o con fev < % del valore predetto. • il bal è metodica diagnostica fondamentale nelle pneumopatie diffuse in soggetti immunodepressi • il bal può essere diagnostico nelle seguenti patologie (con percentuali diverse di rendimento diagnostico):polmonite da pneumocystis jiroveci,tubercolosi,proteinosi alveolare,polmonite lipoidea esogena,emorragia alveolare,polmonite eosinofila,granulomatosi a cellule di langerhans,malt linfoma polmonare primitivo,linfangite carcinomatosa o metastasi polmonari disseminate e altre patologie rare (microlitiasi,sindrome da gaucer o di nieman pick) • il bal può essere diagnostico nelle seguenti patologie (con percentuali diverse di rendimento diagnostico):polmoniti infettive (in cui l'agente causale possa avere teoricamente anche il ruolo di semplice commensale come polmoniti virali,micobatteriosi, vap,etc.),polmonite eosinofila acuta,danno alveolare diffuso (dad),capillarite,sarcoidosi,polmonite in via di organizzazione criptogenetica,embolia grassosa. • il bal è procedura necessaria nell'iter diagnostico delle seguenti patologie:ipf,nsip,rb-ild/dip; lip; polmoniti da farmaci e/o radiazioni;lingangioleiomiomatosi;ipertensione polmonare (malattia veno-occlusiva);altre malattie polmonari rare. • il bal è particolarmente utile come elemento diagnostico nelle patologie polmonari con pattern hrct di tipo alveolare e/o ground-glass. • il bal e la biopsia transbronchiale devono essere eseguiti comunque durante la medesima indagine broncoscopia in caso di patologia polmonare diffusa:quando il pattern è ground glass.negli altri casi non vi sono in letteratura dati dirimenti. • il bal deve essere analizzato da laboratori specializzati. • il bal non ha valore nel monitoraggio delle patologie polmonari diffuse. segue capitolo endoscopia bronchiale diagnostica dell'adulto tabella . continua • la tblb può essere considerata sempre di più una procedura di prima scelta nei pazienti immunocompetenti affetti da pneumopatie infiltrative diffuse in particolare quando si riesca ad ottenere un numero elevato di frammenti ed utilizzando pinze di più grandi dimensioni.l'utilizzo del broncoscopio rigido può essere di ausilio per queste finalità. • i quadri hrct compatibili con un elevato rendimento diagnostico della procedura tblb sono:consolidazioni,ground-glass,treein-bud,nodulazioni,ispessimento dei setti interlobulari (pattern "perilinfatico") addensamenti escavati,presenza del "bronchus sign" . • l'uso della radioscopia,anche se è tuttora discusso,può essere indicata di routine durante l'esecuzione della tblb attraverso il broncoscopio flessibile/rigido. • l'analisi di frammenti bioptici può fornire elementi morfologici di per sé diagnostici (linfangite carcinomatosa,micobatteriosi e altre infezioni quando siano riconoscibili nel tessuto i microrganismi in causa,granulomatosi a cellule di langerhans,linfangioleiomiomatosi,proteinosi alveolare,etc.) o mettere in evidenzi patterns non specifici che vanno messi in correlazione con le ipotesi diagnostiche formulabili in base ai dati clinici e radiologici (organizzazione endoalveolare,granulomi nonnecrotizzanti,polmonite interstiziale cellulata,bronchiolite respiratoria,etc.). • la dimensione del nodulo polmonare (in particolare se questo è < cm) rappresenta il fattore limitante del rendimento diagnostico della procedura tblb in corso di fibrobroncoscopia.tuttavia l'ausilio dell'ecografia per via endoscopica (ebus) migliora sensibilmente il rendimento diagnostico della tblb in presenza di un nodulo polmonare periferico con diametro inferiore a cm.pertanto tale ausilio può essere suggerito. • al fine di una soddisfacente 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- journal: pediatr pulmonol doi: . /ppul. sha: doc_id: cord_uid: wehstffa chronic cough is a common complaint in children and its relationship with asthma is controversial. the aim of the present study was to determine the pattern of airway inflammation in atopic and nonatopic children with chronic cough, and to investigate whether atopy is a predictive factor for eosinophilic inflammation in cough. bronchoalveolar lavage (bal; three aliquots of ml/kg saline) was performed in the right middle lobe of ( atopic and nonatopic) children with persistent cough ( females, males), mean age . years (range: – ). atopy was defined as an elevated total serum ige or a positive rast test. both atopic and nonatopic children with persistent cough had an increase in total cells/ml in bal (atopic: median × ( ), range: – ; nonatopic: median × ( ), range: – ) compared to nonatopic controls (median × ( ), range – ). the increases were mainly in neutrophils (atopic: median %, range . – . %; nonatopic: median %, range . – . %) compared to controls (median . %, range . – . %; atopics vs. controls, p < . ). there were no significant increases in eosinophils, lymphocytes, epithelial cells, or mast cells. eosinophils were elevated in only / atopic and none of the nonatopic children. the increased percentage of neutrophils in the bal fluid of atopic and nonatopic children with persistent cough could be due to an underlying inflammatory process driving the cough, or even conceivably, due to the effect of coughing itself. in this highly selected series, the absence of eosinophilic inflammation in the majority suggests that most would be predicted not to respond to inhaled corticosteroid therapy. this study underscores the need to be cautious about treating coughing children with inhaled corticosteroids, even in the context of a tertiary referral practice. pediatr pulmonol. ; : – . © wiley‐liss, inc. cough is a primary defense mechanism that functions to protect the airways, clearing irritants and mucus. cough can be classified into acute and chronic. chronic cough can be defined as a cough persisting for at least weeks however this definition is arguable and others have regarded duration more than weeks as chronic cough. chronic cough is a common complaint in children, with a wide differential diagnosis. in a community setting, most children with chronic cough do not have asthma, although recent diagnostic fashion has been to assume that all chronic cough is due to asthma, and treat it with inhaled corticosteroids. thus this common condition may frequently be overdiagnosed as asthma, , and harmful medication may be continued for a long period of time without benefit for these children. the relationship between chronic cough and asthma is controversial. although cough variant asthma is recognized as a precursor of bronchial asthma, it is not known whether chronic cough is also a precursor of asthma. atopic patients with chronic cough due to cough variant asthma are thought to have airway inflammation similar to atopic patients with asthma, whose bronchoalveolar lavage (bal) fluid contains eosinophils and mast cells. in adults, eosinophils are believed to be a key effector cell in asthma. some studies , have provided evidence of eosinophilic inflammation in the bal of children with asthma. marguet et al. demonstrated that the bal profile in asthma is clearly characterized by a high proportion of eosinophils, which is independent of the severity of the disease and treatment with inhaled steroids. most previous studies have studied milder forms of cough, and there is a paucity of data in a tertiary context, where one might expect there to be a greater likelihood of finding pathology. , the aim of the present study was therefore to determine the pattern of inflammation in atopic and nonatopic children with chronic cough, and to investigate whether atopy is a predictive factor for eosinophilic inflammation in this context. we studied ( atopic and nonatopic) children ( females and males) with a mean age of . years (range - years) seen at the pediatric outpatient clinic of the royal brompton hospital with a history of chronic cough (table ) . all had coughed for at least months. fifteen had used inhaled corticosteroids, with no evidence of benefit. fifty percent of these children had cough for periods longer than year, and / ( %) have nonproductive (dry) cough. inclusion criteria were chronic cough (cough for more than weeks), absence of respiratory tract infection in the preceding days, absence of alternative diagnosis such as cystic fibrosis, immunodeficiency, sinusitis and ciliary dyskinesia and agreement of parents after informed consent signature. routine tests performed in these children included chest x-ray and high resolution ct scan, full blood count, serum immunoglobulins, sweat test, nasal brushing for ciliary motility and structure studies, -hr ph study. spirometry and exhaled nitric oxide were performed in those older than years old, and evaluation by an otorynolaryngologist performed when indicated. six children (three atopic and three nonatopic) were receiving inhaled steroids. atopy was defined as a positive radioallergosorbent (rast) test (eight children) or elevated total serum ige (compared to age appropriate ige ranges, three children). all children with chronic cough underwent bronchoscopy for clinical purposes. bal was also performed on five children without chronic cough (control group) who underwent bronchoscopy for other clinical reasons, (three children underwent bronchoscopy for investigation of vascular and structural malformation and two for stridor). all were nonatopic and had no history of asthma. none of the patients had a clinical history of respiratory infection in the weeks prior to bronchoscopy, albeit / patients reported use of antibiotics for other reasons in this period (one received azythromycin and the other amoxicillin-clavulanate). alternative diagnoses such as sinusitis, immunodeficiency, cystic fibrosis, tuberculosis, and primary ciliary dyskinesia were excluded in all patients by conventional tests. chest radiographs showed opacities suggestive of atelectasis in only / children, but none of them had evidence of bronchiectasis on ct scanning. eighteen of the patients with chronic cough had also undergone a -hr esophageal ph test for gastroesophageal reflux (ger) as part of a separate study. the test was defined as positive if the ph was less than for greater than % of the -hr study period. the study was approved by the ethics committee of the royal brompton hospital, and written informed consent was obtained from the parents of the subjects, and age-appropriate assent from the children. the same bal protocol was used in all patients. the bronchoscopy was performed under general anesthesia using a laryngeal mask airway, or via a facemask with careful attention not to use suction until the bronchoscope had passed the vocal cords; the protected brush method was not utilized. bal was performed in the right middle lobe in all patients and controls using three aliquots of ml/kg saline ( table ) . bal fluid was centrifuged at , rpm ( g) for min at c. the supernatant was removed and the cell pellet was resuspended in ml of minimal essential medium (mem) containing hepes buffer (invitrogen, london, uk). total bal cell counts were obtained using an improved neubauer counting chamber and the results are expressed as number of cells per ml. cytocentrifuge preparations were made using ml aliquots of a . Â cells/ml suspension. after air-drying, the preparations were stained with may-grünwald giemsa to make differential cell counts. the percentages of each cell type were determined by counting cells. a bal sample was sent for microbiological analysis. quantitative cultures were performed in selective media in the routine microbiological laboratory, and a bacterial count ! cfu/ml was considered positive. additionally, direct immunofluorescence was performed in bal samples using specific monoclonal antibodies against respiratory syncytial virus, influenza a and b, parainfluenza , , and , and adenovirus. the data were analyzed using nonparametric tests. the mann-whitney test was used to test differences between unpaired groups of quantitative data and differences were considered to be significant when p . . bal results are summarized in table . recovery of bal fluid was similar in atopic (median: %, range: - %) and nonatopic (median: %, range: - %) patients, but lower than in the control group (median: %, range: - %), albeit the difference was not statistically significant. a nonsignificant increase in the number of total cells per ml of bal fluid was observed in both atopic (median: Â , range: - Â ) and nonatopic (median: Â , range: - Â ) children with chronic cough when compared to controls (median: Â , range: - Â ). the increases in total cells were mainly due to increases in neutrophils in both the atopic (median: %, range: . - . %) and nonatopic (median: %, range: - %) children with chronic cough compared to controls (median: . %, range: . - . %). in total, of the nonatopic and of the atopic children with chronic cough had neutrophil percentage counts above the upper limit of % for the controls. however, the percentage of neutrophils was significantly higher only in the atopic children compared to controls, p ¼ . (table and fig. ). microbiological analysis of the bal samples was negative for bacteria and viruses in all cases. there were no significant differences in the number or percentages of lymphocytes, epithelial cells or mast cells. the percentage of bal eosinophils was elevated (> %) in five of the atopic children, although there was no increase in eosinophils in the nonatopic group (fig. ) . however, the slightly higher percentages of eosinophils in the atopic group did not reach significance compared with the other two groups (fig. ) . of the children with chronic cough who underwent -hr ph testing for ger, nine were positive (four atopic and five nonatopic). median bal neutrophil percentages were higher in the nine patients with a positive ger (median: %, range: . - . %) compared to the nine with a negative ger (median: . %, range: - %), but the difference was not statistically significant (p ¼ . , fig. ). seven children with ger showed more than % of neutrophils in the bal, although this finding was present in five children without ger. lipid laden macrophages were measured in of children. of the nine with a positive ph study, only three had an index greater than . chronic cough, defined as a cough persisting for at least weeks, is a common symptom in childhood. there is little information about the pathophysiology and the airway cellularity in children with chronic cough. [ ] [ ] [ ] we analyzed the inflammatory cell profile of bal fluid from atopic, nonatopic and normal children with chronic cough. the population of coughing children is highly selected, having been deemed sufficiently severe to warrant referral and investigation in a tertiary center; none had a diagnosis of a specific chronic chest disease such as bronchiectasis prior to diagnosis. the control children were matched for age as far as possible, but since ethically we could only perform a bronchoscopy in a child in whom it was clinically indicated, this group is necessarily limited. nonetheless, our control data are similar to those of others who were able to study larger groups. in the present study, the majority of the atopic children and nearly half of the atopic children with chronic cough had an increase in the percentage of bal neutrophils when compared to controls; but even in this selected group, eosinophilia was absent in the nonatopic coughers, and present in less than % even of atopic children with cough. an important issue is the upper limit of normal of airway neutrophilia in children. because healthy children cannot be studied for ethical reasons, pediatric bal reference values are difficult to obtain. it is probable that the differential cytology in children is similar to that observed in healthy adults. although bal neutrophil of % is the upper limit of normal reported by middula et al. (range - %) and ratjen et al. ( - %) in five other studies the median normal value for neutrophils is given as . % [ ] [ ] [ ] [ ] [ ] hence in our study, the median value of bal neutrophils ( %) and the values in both the non atopic group ( . - . %) and atopic group ( . - %) were appreciably higher than the values reported in normal children. the mechanism of the neutrophilia is unclear. one of the most common causes of chronic cough in children is recurrent viral infection, with some viral infections causing prolonged periods of cough. an increased percentage of bal neutrophils may be due to an underlying inflammatory process such as occult persistent infections, caused by bacteria or viruses. , [ ] [ ] [ ] in a recent cohort study published by marchant et al., a standardized pathway of investigation of chronic cough in children revealed that protracted bacterial bronchitis diagnosed by bal was the most common diagnosis among children, with significant higher neutrophil levels on bal samples. none of our patients had clinical evidence of a respiratory infection in the weeks prior to bronchoscopy, and microbiological analysis of the bal samples was negative for bacteria and viruses. however, we cannot rule out the possibility of a viral infection in these children, because molecular biology methods were not utilized and may improve viral detection, while represent the main tool for detection of some significant respiratory viruses such as rhinovirus, coronavirus, and human metapneumovirus. pertussis, another potential pathogen quoted as a substantial cause of prolonged cough in school age children would be an unlikely etiology once % of our children had cough for periods longer than year (range - years). however, we cannot exclude the possibility of a pertussis infection, because we did not do serology, and children who have been partial vaccinated may have prolonged dry cough without the other classical features of whooping cough. it is also possible that we have underdiagnosed other infections in this group. we defined a positive culture a ! colony forming units per milliliter of balf because the bronchoscopy was performed using a laryngeal mask airway, or via a facemask with careful attention not to use suction until the bronchoscope had passed the vocal cords; the protected brush method was not utilized. the ers task force defined this cut off for non protected bal specimens. baker et al. also recommended that the cut off level of cfu/ml for bal is appropriate when pneumonia is suspected and a cut off level of cfu/ml for bal is appropriate if the probability of disease is low. lower cut off levels have indeed been used by some investigators when a protected brush is employed (> cfu/ml) or with suspected ventilator-associated pneumonia (> cfu/ml). we acknowledge the difficulty of establishing a diagnostic threshold for quantitative culture on bal for bronchitis in children (as opposed to pneumonia). the second potential source of underdiagnosis is sampling error; although there are data on differences between bal in the context of cf we know of no such data on the chances of missing positive cultures in the context of bacterial bronchitis. another potential cause of chronic cough is ger. irwin et al. demonstrated that chronic cough can be the sole manifestation of ger in adults. controversy exists regarding the presence of inflammatory markers in the bal fluid of children with ger, but some studies have shown that ger may increase the percentage of bal neutrophils. in the present study, of children ( atopic and nonatopic) had a positive -hr ph test (> % of the study period with ph < ). seven children (four atopic and three nonatopic) who had a positive ph test also had an increased percentage of bal neutrophils. whether or not ger is associated with or is the cause of airway inflammation in children with chronic cough cannot be determined from these cross-sectional data (fig. ) . a higher percentage of bal neutrophils in patients with chronic cough may also be explained by exposure to environmental tobacco. unfortunately, in the present study we did not actively investigate exposure of the children to tobacco smoke using urine or salivary cotinine. conceivably, the mechanical effect of coughing could itself cause airway neutrophilia; to our knowledge, this has never been tested, and it is difficult to believe that such profound neutrophilia as reported here could be caused by the mechanical effects of cough alone. asthma is usually characterized by wheeze and dyspnoea, and there is controversy as to whether pure cough variant asthma exists. [ ] [ ] [ ] [ ] [ ] cough variant asthma is usually diagnosed in a child with persistent cough who has airway hyperresponsiveness and a good response to antiasthma medication, with relapse of symptoms when the medication is stopped. atopic children with chronic cough due to cough variant asthma would be predicted to show airway inflammation similar to atopic children with asthma, whose bal fluid contains eosinophils and mast cells. , our results are consistent with the findings of other investigators, who found that chronic cough was rarely associated with eosinophilia in bal or induced sputum. , , , furthermore, data from the tucson cohort study have shown that cough without wheezing had a more favorable prognosis than cough with recurrent wheezing, suggesting that chronic cough differs from asthma in several aspects and may have a different pathophysiology. in the present study, atopy did not predict eosinophilic airway inflammation, suggesting that only a minority of these children have eosinophilic asthmatic-type airway inflammation. a review of the use of medications in children with persistent cough has demonstrated an overdiagnosis of asthma and overuse of asthma treatments, with a potential risk of side effects in children with chronic cough. , one australian study concluded that, although children with chronic cough were similar to asymptomatic children in terms of atopic status, family history and respiratory morbidity, the rates of asthma diagnosis and use of asthma medication was higher in these children. in the present study, ( atopic and nonatopic) of the children were using inhaled steroids. we accept that it would have been more rigorous to stop this medication prior to bronchoscopy, but this was not thought to be ethical. one child had an increase in bal eosinophils, despite being prescribed inhaled steroids, although no increase in bal eosinophils was observed in the others. it is possible that in these children inhaled corticosteroids might have suppressed bal eosinophils. none of the patients reported any improvement in symptoms related to inhaled steroid therapy, and indeed, all were still symptomatic at the time of bronchoscopy despite this treatment. we do not have any information on adherence to therapy. however, as a group, children with chronic cough did not exhibit asthmatic-type airway inflammation, underscoring the need for caution in the diagnosis of cough variant asthma, even in patients with severe symptoms investigated in a tertiary center. in conclusion, the evaluation of children with persistent cough demonstrated that atopic and nonatopic children with chronic cough frequently have an increased percentage of bal neutrophils, the cause of which is not known, but which may be due to an underlying inflammatory process. further studies are needed to determine the mechanism of neutrophilia in children with chronic cough. atopy did not predict eosinophilic airway inflammation in children with chronic cough. few of the atopic children, and none of the nonatopics with persistent cough had asthmatic-type eosinophilic airway inflammation, and thus would be predicted not to respond to inhaled corticosteroid therapy. chronic cough in children all that cough is not asthma state of art: cough, cough receptors and asthma in children comparison of atopic cough with cough variant asthma: is atopy cough a precursor of asthma? eosinophilic tracheobronchitis and airway hypersensitivity in chronic nonproductive cough correlation of bronchial eosinophils and mast cell activation with bronchial hyperresponsiveness in children with asthma inflammatory mediators in bronchoalveolar lavage samples for children with and without asthma bronchoalveolar lavage cell profiles in children with asthma, infantile wheeze, chronic cough or cystic fibrosis evaluation and outcome of young children with chronic cough cough quality in children: a comparison of subjectss bronchoscopic findings gastroesophageal reflux and inflammation in bronchoalveolar lavage in children (abstract) airway inflammation in nonasthmatic subjects with chronic cough evaluation and outcome of young children with chronic cough cough and reflux esophagitis in children: their co existence and airway cellularity bronchoalveolar lavage in children. ers task force on bronchoalveolar lavage in children bronchoalveolar lavage cellularity bronchoalveolar lavage studies in children without parechymal lung disease: cellular constituents and protein levels lymphocytes subsets in bronchoalveolar lavage fluid of children without bronchopulmonary disease investigating paediatric airways by non lavage: normal cellular data bronchoalveolar lavage cellularity in healthy children a controlled study of differential cytology and cytokine expression profiles by alveolar cells in paediatric sarcoidosis analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection rhinovirus colds in healthy and asthmatic subjects: similar changes in upper and lower airways persistent and latent viral infections in the pathology of asthma chronic cough in children: bronchoalveolar lavage findings recent developments in pertussis belohradsky bh and the munich vaccine study group. clinical and epidemiological picture of b pertussis and b parapertussis infection after introduction of accelular vaccine discussion making in nosocomial pneumonia. an analytic approach to the interpretation of quantitative bronchoscopic cultures blind protected specimen brush and bronchoalveolar lavage in ventilated children a bronchoscopic scoring system for airway secretions-airway cellularity and microbiological validation interlobar differences in bronchoalveolar lavage fluid from children with cystic fibrosis chronic cough as the sole presenting manifestation of gastroesophageal reflux lipid laden macrophage index and inflammation in bronchoalveolar lavage fluids in children bronchoalveolar lavage and esophageal ph monitoring data in children with difficult to treat respiratory symptoms increased neutrophils and cytokines, tnf and il in induced sputum of non-asthmatic patients with chronic dry cough chronic cough as the sole presenting manifestation of asthma cough variant asthma: a review of the clinical literature cough-but is it asthma? isolated cough-probably not asthma clinical significance of cough and wheeze in the diagnose of asthma bronchoalveolar lavage findings suggest two different forms of childhood asthma airway eosinophilia is associated with wheeze but is uncommon in children with persistent cough and frequent chest colds induced sputum: comparison of postinfections cough with allergic asthma in children recurrent cough in childhood and its relation with asthma persistent cough in children and the overuse of medication cough in children clinical significance of cough and wheeze in the diagnosis of asthma persistent cough: is it asthma? the authors thank dr. claudio leone for the technical assistance. this study was supported by royal brompton hospital charitable fund (no. b ; to plh). key: cord- -ffulkqrf authors: versluys, anne birgitta; van der ent, korstiaan; boelens, jaap j.; wolfs, tom; de jong, pim; bierings, marc b. title: high diagnostic yield of dedicated pulmonary screening before hematopoietic cell transplantation in children date: - - journal: biol blood marrow transplant doi: . /j.bbmt. . . sha: doc_id: cord_uid: ffulkqrf pulmonary complications are an important cause for treatment-related morbidity and mortality in hematopoietic cell transplantation (hct) in children. the aim of this study was to investigate the yield of our pre-hct pulmonary screening program. we also describe our management guidelines based on these findings and correlate them with symptomatic lung injury after hct. since , all patients undergo a dedicated pulmonary screening consisting of pulmonary function test (pft), chest high-resolution computed tomography (hrct), and bronchial alveolar lavage (bal) before hct. we systematically evaluated the yield during the first years of our screening program. we included consecutive children. in % of patients, abnormalities were found. in % of patients, or more pft results were < % of normal. chest hrct showed abnormalities in %; % of these abnormalities were considered “clinically significant.” bal was abnormal in % of patients; respiratory viruses (pcr) were found in patients, fungi (antigen or culture) in , and bacteria (culture) in . all screening tests contributed separately to clinically relevant information regarding pulmonary status in these pre-hct children. in patients ( %), screening results had diagnostic and/or therapeutic implications. we found an association between pre-sct hrct findings and lung injury after transplantation. pre-hct screening with the combination of modalities, reflecting different domains of respiratory status (function, structure, and microbial colonization), reveals important abnormalities in a substantial number of patients. whether this improves patient outcome requires further investigation. hematopoietic cell transplantation (hct) is a curative treatment for various diseases. pulmonary complications, both infectious and noninfectious, are frequently seen in patients undergoing hct. in children, the incidence of pulmonary complications varies from % to % and is associated with a significantly increased risk for mortality [ ] [ ] [ ] . because of the risk of life-threatening complications of the procedure, patients are routinely screened for hct eligibility. lung screening can potentially impact selection of hct patients as well as affect preemptive treatment and prognosis. invasive fungal infections (ifi) are an important cause of morbidity and mortality during hct. diagnostic imaging, culturing pathogens, and antigen detection can be helpful to identify patients at high risk for ifi, which may guide therapy [ ] . also, respiratory viruses (rv) may have impact on the overall survival of hct, either directly as a cause of pneumonitis in the severe immune-compromised patient or indirectly by triggering allo-immunity in the setting of allogeneic transplantation [ ] . in , we implemented extensive pre-hct lung screening, which includes pulmonary function test (pft), chest high-resolution computed tomography (hrct), and bronchial alveolar lavage (bal) in all patients. here, we evaluate the yield of such an extensive pulmonary screening biology of blood and marrow transplantation j o u r n a l h o m e p a g e : w w w . b b m t . o r g program and describe our treatment guidelines according to these findings as well as the outcome of patients. all consecutive pediatric patients undergoing a first allogeneic hct in our center between january and august were included. patients were enrolled in the hct research protocol after providing written informed consent for data collection and analysis, according to national ethical regulations (ethical commission number / and / k). patient characteristics (age, gender, underlying disease), clinical symptoms, results of pulmonary screening tests, and occurrence of symptomatic lung disease after hct was registered. standard pre-hct pulmonary screening is performed in the week before transplantation and consists of a pft, hrct scan, and bal. pft includes spirometry, whole body plethysmography, and measurement of carbon monoxide diffusion capacity. measurements are performed in children aged years and older, according to american thoracic society/ european respiratory society criteria, using calibrated pneumotachometer systems (jaeger, hochberg, germany). values are expressed as percentage of predicted values for age, race, sex, and height-matched controls (the utrecht data set, koopman [ ] ). forced expiratory volume in second, forced vital capacity, total lung capacity, and lung diffusion capacity for co, corrected for hemoglobin and alveolar volume < % of predicted values are considered to be abnormal. residual volume of > % of total lung capacity is considered to be abnormal and suggestive for trapped air. hrct scans are acquired using a -detector row scanner (philips medical systems, best, netherlands). for infants and young children, scans are obtained at -cm h o pressure (inspiration) and -cm h o pressure (expiration). for older children, who were able to cooperate with breath hold instruction, scans were obtained at full inspiration and at end of exhalation. inspiration images are obtained using fixed kvp and to mas (depending on bodyweight). for expiration images, we used kvp and mas. acquisition was volumetric thin-slice for both inspiratory and expiratory computed tomography. all hrct scans were assessed by a pediatric radiologist. fleischner society terms for thoracic imaging were used [ ] . all abnormalities, as stated in the radiology report, were registered. those abnormalities with clinical implications, such as antimicrobial treatment, guided lung biopsy or diuretics, were defined as clinically significant. bal was performed under general anesthesia. bal fluid was cultured and processed in accordance with standard microbiological procedures. galactomannan (gm) tests are performed using biorad platelia aspergillus eia. any positive culture or gm levels > . was considered to be abnormal. nucleic acids are extracted using the total nucleic acid protocol with the magna pure lc nucleic acid isolation system (roche diagnostics, basel, switzerland). for detection of rna-viruses cdna is synthesized by using multiscribe reverse transcriptase and random hexamers (applied biosystems, foster city, ca). detection of viral and atypical pathogens was performed in parallel, using real-time pcr assays specific for the following viruses: bocavirus, human herpesviruse , respiratory syncytial virus, influenzavirus a and b, parainfluenzavirus to , rhinoviruses, adenoviruses, human coronavirus oc , nl and e, human metapneumovirus, and mycoplasma pneumoniae. real-time pcr procedures were performed as described previously [ ] . any positive pcr is considered to be abnormal. the total costs for the pulmonary screening were approximately euro. chest hrct costs euro, rv panel pcr , bacterial cultures euro, gm euro, pft (complete) cost euro. for further analysis, patients were classified according to their risk for pre-hct pulmonary problems, based on underlying disease, immune competence, infection risk, and pretreatment with potentially lung-toxic therapy. we distinguished groups of patients: those with an inherited immune deficiency, those with a malignant disease and chemotherapy before transplantation, and those with inborn errors of metabolism, mild bone marrow failure, and malignancies without chemotherapeutic treatment. antibiotic prophylaxis involved daily ciprofloxacin and fluconazole, from the start of conditioning until the resolution of neutropenia. additional prophylaxis against streptococcus viridans was given with cefazoline in the mucositis phase. empiric antibiotic treatment for febrile neutropenia included vancomycin and ceftazidime. pneumocystis jeroveci pneumonia prophylaxis was started from month after transplantation as cotrimoxazole times a week. in case of positive serology for herpes simplex virus in all patients, and in case of positive serology for varicella zoster virus in cord blood transplantation recipients, prophylaxis with aciclovir was given. no other antiviral prophylaxis was given. in patients at high risk for ifi, according to our protocol, based on pretreatment, duration of neutropenia, and history of fungal infection, aspergillus prophylaxis was given with daily voriconazole or twice weekly amphotericin b. patients with severely impaired pft (< % of normal) were considered to have an unacceptable high risk for treatment-related mortality and were excluded for hct. patients with rv from bal were considered to have a high risk for alloimmune-mediated lung syndromes. in elective hct procedures, hct was postponed until the rv was cleared. in other casesdwhen the underlying disease did not allow treatment delaydtapering of immune suppression after hct was adjusted to prevent alloimmune-mediated lung syndromes. in cases with probable fungal disease (positive cultures or gm from bal), antifungal treatment was considered. patients with positive bacterial cultures from bal were not treated, unless pulmonary symptoms developed. bacterial culture results guide the choice of empirical antibiotic treatment for neutropenic fever after hct. in patients with nodular lesions on hrct, lung biopsy was considered to identify the possible infectious cause and antimicrobial resistance pattern. in patients with possible or proven ifi based on bal findings, biopsy results, or hrct findings, antifungal treatment was started and granulocyte transfusions or haploidentical stem cell support (combined with cord blood grafts) were considered. calculation of mean values and standard deviation was done for pfts. comparing the results with predicted values for age, race, sex, and heightmatched controls was done using t-test (test value %). comparison of the means between the different disease groups was done using anova. the chi-square test was used for comparison of proportions between or more groups. differences with a p value of < . were considered statistically significant. associations between pre-hct pulmonary screening findings and clinically manifested lung injury after hct were analyzed using cox proportional hazard models. dichotomous outcomes were used as dependent variables. univariate predictors with a p value of < . were used for multivariate analysis. all statistics were done using spss . we included consecutive children receiving a first allogeneic hct. apart from mild upper respiratory tract symptoms in some, all patients were asymptomatic for lung disease at the time of pre-hct screening. patient characteristics are shown in table . in patients, no pre-sct lung screening was performed at all; of them were under months of age. in patients ( %), all planned screening modalities could be performed. in children, only some of the tests were done either for logistic reasons (n ¼ ) or because screening bal was the only reason for general anesthesia, which was then considered a disproportional invasive procedure (n ¼ ). in children above the age of , pft was not feasible because of developmental delay. in patient, hrct was omitted because of the risk of irradiation damage related to underlying disease (fanconi anemia). pft were performed in patients. this was % of all patients in whom we were able to perform these tests, according to age and developmental level. results are shown in table . we found pft patterns of restrictive and obstructive lung disease as well as diffusion abnormalities with forced expiratory volume in second, mean . % (sd . ); forced vital capacity, mean . % (sd . ); total lung capacity, % (sd . ); and lung diffusion capacity for co, corrected for hemoglobin and alveolar volume, . % (sd . ). compared with the reference population with values of % (sd ), all differences were statistically significant; with p values of < . . there was no difference in abnormalities in pft between the different disease categories, see table . chest hrct was performed in ( %) patients. in patients ( %), abnormalities were seen; in of patients ( %) these findings were "new" findings. in the group of patients without pretreatment with chemotherapy or immune deficiency, the incidence of hrct abnormalities before hct was significantly lower than in the other patients ( figure ). in patients ( %), clinically significant abnormalities were found. four ( %) had lesions suspect for fungus. fourteen patients ( %) showed other abnormalities, including bronchiectasis, pleural effusion, consolidations, and aspecific nodules > cm. these were new findings in of patients ( %). most clinically significant abnormalities were found in the subgroup of patients with immune deficiencies, but this did not reach statistical significance ( figure ). bal was performed in ( %) patients. unfortunately, for logistic reasons, it was not always possible to do all microbial tests. overall, in % of tested patients, or more of the microbial tests were positive. positive pcr for rv was found in ( %) of tested patients. rhinovirus was the most frequently detected virus (table ). in ( %) patients, we found microbial evidence of fungal colonization either with positive cultures or gm. in only patients, a positive gm corresponded with a positive culture for aspergillus; in patient with a positive aspergillus culture, gm from bal was negative. the positive findings for the whole cohort are shown in table . in patients under years of age, the incidences of bal abnormalities in general, rv positivity, and bacteria positivity were significant higher than in older patients (p values of . , . , and . respectively). figure shows the yield of all screening tests. abnormalities were found in tests of % of patients. we found abnormalities in radiological tests among patients with normal and abnormal pfts, as well as positive microbial test results in patients with normal pfts and normal hrct. in patients, the screening outcome had implications, such as guided bal/lung biopsy (n ¼ ), change in antifungal treatment/prophylaxis (n ¼ ), granulocyte transfusions (n ¼ ), addition of haploidentical stem cells (n ¼ ), postponement of hct (n ¼ ), or guided tapering of immune suppressive agents (n ¼ ). these interventions overlapped in some patients. in patients, the pre-hct hrct showed new or progressive signs of infiltrative fungal infection. antifungal therapy was intensified in based on resistance pattern of the cultured pathogen. in patients, we postponed hct. in cord blood transplant recipients, we added haploidentical cd þ cells from a family donor for early myeloid support, and in patients we gave granulocyte transfusions during the period of neutropenia. one patient had graft rejection and showed fatal progression of aspergillus infection in prolonged neutropenia. the others did not show progression of infection, and therapy could be stopped safely after engraftment. no patient with rv or bacteria isolated showed progression of pulmonary infection during hct. none of the patients with isolated positive findings for fungus from bal, of whom the majority received intensification of fungal prophylaxis/ treatment, developed pulmonary fungal disease. cox regression analysis did not show a relation between pre-hct screening findings in bal or pft and symptomatic lung injury after hct. a clinically significant abnormality on chest hrct before hct, however, was a predictor for the development of immune-mediated lung injury after hct (hazard ratio, . ; % confidence interval, . to . ; p ¼ . ). our study in pediatric patients shows that pulmonary screening before hct with pft, hrct, and bal is feasible. we could perform all the tests in the majority of patients ( %). abnormalities were found in % of patients. in % of * galactomannan in bal > . ¼ positive. y there was overlap between fungal culture results and galactomannan findings in bal ( of aspergillus-positive patients were also galactomannan positive, patient with candida and with penicillium also had positive gm) so patients were considered positive for fungus. patients, these abnormalities led to supportive/preemptive treatment according to guidelines. only in patients with clinically significant chest hrct, abnormalities a higher incidence of lung-injury was noted after hct. although not negligible, the costs seem justified in relation to the findings. it is well known that pulmonary function declines early after hct [ , ] , and some studies have shown a continuous decline without reaching a plateau during prolonged followup [ ] . several studies have demonstrated that impaired pft before transplantation increases the risk for post-transplantation lung complications and mortality [ , , [ ] [ ] [ ] [ ] . possible explanations for these observations are that patients can have such marginal lung reserve capacity, endangering a period of critical illness and/or further lung toxic events. also, in patients with pre-existing lung injury, this organ may be at increased risk for allo-immune phenomena, such as graft-versus-host disease. we evaluated the yield of hrct scanning. omitting hrct from our screening in this cohort would have missed ( %) children with abnormalities, including of the with infiltrative lesions suspect for fungus. on the other hand, hrct leads to radiation exposure and may require general anesthesia in children and, therefore, deserves critical appraisal. the relevance of abnormal findings on hrct are a matter of debate. in the radiological reports in this study, abnormalities were described in % of patients. because the severity of the reported abnormalities varied considerably, we chose to take into account those hrct findings which had "significant clinical meaning" at time of transplantation, such as consolidations requiring antibiotic or antifungal therapy, bronchiectasis as a risk factor for infections warranting change in prophylaxis, or pleural effusions requiring diuretics. in most patients, a plain chest x-ray was available but showed abnormalities in only %, and, of note, did not show any abnormalities in the patients with signs of invasive fungal infection on hrct (data not shown). the yield of bal procedures was high in our study. omitting bal would have missed ( %) patients with fungal colonization and ( %) with rv. all these patients had normal hrct scans and no significant pulmonary symptoms. figure illustrates that all tests contribute separately to information regarding pulmonary status in pre-hct children. this argues that all these screening modalities, reflecting different domains of respiratory status (function, structure, and microbial colonization), should be done in all pediatric pre-hct patients, if a sensitive pre-hct screening for pulmonary pathology is desired. the impact of the finding and the invasiveness of the test should guide clinicians in decision-making on whether or not to perform all tests. as far as we know, this is the first report on such comprehensive pulmonary screening with pft, hrct, and bal in a large cohort of children before hct. we have shown considerable decrease in pulmonary function, a significant amount of clinical important hrct findings, and high prevalence of infectious agents. this is most likely because of underlying disease, pretreatment with chemotherapy, and the age distribution of our patients. in this cohort of patients, there is a significant association between clinically significant hrct findings before hct and lung injury after hct. the findings in bal and pft were not related with outcome. this might be due to small numbers. we might also conclude that with current treatment strategies for this group of pulmonary compromised patients, we manage to have comparable outcome. we conclude that this screening protocol is feasible and provides important information for risk classification with therapeutic consequences. we would advocate all screening methods, as they all contribute separately. prospective studies are needed to further identify the importance of baseline abnormalities in the risk for pulmonary complications and treatment-related mortality and whether outcome is improved by using intensive screening. financial disclosure statement: there are no items to disclose. conflict of interest statement: there are no conflicts of interest to report. authorship statement: a.b.v. had full access to all data in the study and takes responsibility for integrity of data and analysis. c.k.e., j.j.b., t.w., p.d.j., and m.b. contributed equally and substantially to study design, interpretation of data, and writing of the manuscript. lung function, pulmonary complications and mortality after allogeneic blood and marrow transplantation in children lung function and late pulmonary complications among survivors of hematopoietic stem cell transplantation during childhood natural history of pulmonary complications in children after bone marrow transplantation diagnosis of invasive fungal infections in hematology and oncology-guidelines from the infectious disease working party in haematology and oncology of the german society for haematology and oncology (agiho) strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes reference values for paediatric pulmonary function testing: the utrecht dataset fleischner society: glossary of terms for thoracic imaging 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 pulmonary dysfunction in survivors of childhood hematological malignancies after allogeneic hematopoietic stem cell transplantation lung function after allogeneic hematopoietic stem cell transplantation in children: a longitudinal study in a population based cohort pulmonary function testing prior to hematopoietic stem cell transplantation influence of pretransplantation restrictive lung disease on allogeneic hematopoietic cell transplantation outcomes lung function and long-term complications after hematopoietic cell transplant pretransplant lung function is predictive of survival following pediatric bone marrow transplantation key: cord- - rcj tc authors: jia, bei; lovari, robert; miller, heather; metzgar, david; massire, christian; carolan, heather; toleno, donna; d'alessio, franco; rothman, richard; blyn, lawrence b.; zhang, sean x. title: evaluation of a pcr-electrospray ionization mass spectrometry platform for detection and identification of fungal pathogens directly from prospectively collected bronchoalveolar lavage specimens date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . sha: doc_id: cord_uid: rcj tc the incidence of invasive fungal infections is on the rise worldwide due to the growth of the immunocompromised population. we report here the use of a diagnostic assay that utilizes a universal extraction method, broad spectrum pcr amplification and analysis via electrospray ionization mass spectrometry (pcr/esi-ms) to detect and identify more than pathogenic fungi directly from bronchoalveolar lavage (bal) specimens in less than hours. in this study, we describe both analytical and clinical performance of the assay, when run with prospectively collected clinical bal specimens. in patients with probable and possible fungal infections defined by eortc/msg (european organization for research and treatment of cancer/mycoses study group) criteria, the pcr/esi-ms assay demonstrated a sensitivity of . % ( % ci: . – . %) and a specificity of . % ( % ci: . – . %). this data demonstrates the utility of a non-culture based broad fungal targets molecular diagnostic tool for rapid and accurate diagnosis of invasive fungal infections in patients at risk of developing fungal diseases. the diagnosis of fungal infections is clinically challenging and often relies on a combination of clinical, radiologic, and microbiological factors. traditional method of culture-based fungal identification is a time consuming process with some fungi requiring up to weeks of growth before identification can be made. such lengthy turn-aroundtimes do not meet the clinical need for management of fungal infections, which are increasing in frequency given the ever-enlarging immunocompromised patient population, largely driven by widespread use of immune-suppressive and immune-modulatory therapies (harpaz et al., ) . rapid and accurate identification of fungal pathogens is known to impact on the appropriate antifungal drug choice, since fungal pathogens exhibit different antifungal susceptibility profiles (albataineh et al., ; . non-culture-based technologies have been developed to aid clinical laboratories in the rapid and accurate identification of invasive fungi. these technologies include antibody detection, antigen detection of fungal proteins and polysaccharides, proteomic profiling and genomic identification by pcr and dna sequencing (zhang, ; albataineh et al., ; perlin and wiederhold, ) . each technology varies in sensitivity, breadth of coverage, rapidity and ease of use. technologies such as pcr provide rapid and sensitive detections of specific targets e.g. aspergillus species directly from clinical specimens (white et al., ) , but remain limited due to inability to detect the breadth of potentially invasive fungi. pcr/esi-ms is a technology that combines the rapidity and sensitivity of pcr with the breadth of database coverage of more than fungal species. the pcr/esi-ms technology had previously been evaluated for the detection and identification of fungi in both pure culture isolates and retrospectively archived frozen respiratory specimens, and were shown to be sensitive to diverse species representing fungi in all pathogenic clades (massire et al., ; shin et al., ) . unlike these previous studies, our current study determined both analytical and clinical performance of the pcr/esi-ms assay in patient-consented diagnostic microbiology and infectious disease xxx (xxxx) xxx prospectively collected bal samples. for the analytical performance, sensitivity (or limit of detection) and specificity of the pcr/esi-ms assay for fungal detection (that were lack in previous publications) were assessed. for the clinical performance, the pcr/esi-ms method was applied to prospectively collected bal specimens obtained from patients suspected of, or at high risk for, pulmonary fungal infections. in addition, an updated detection platform, capable of handling larger input volumes ( ml vs. ml), with an improved signature database and a more rapid processing time (b hours sample to answer), was used. results were analyzed relative to comparator standard care of mycology laboratory testing, which included culture, biomarker, histopathology, and molecular data, along with clinical data from the same patients. the pcr/esi-ms assay was designed to detect and identify over pathogenic fungi belonging to genera. an additional nonpathogenic fungi which were found to have little or no history of human pathogenicity were included in the assay database and reported under a non-specific fungal detection category. the pre-filled well assay strips contained primers targeted against widely conserved fungal genes in the small and large ribosomal subunits, as well as against more narrowly conserved ribosomal genes found in fungal mitochondria and the beta-tubulin gene. the distribution of this gene across the fungal divisions was shown in supplement fig. . a sixteenth primer was used for to control for extraction and amplification. these primers, pcr cycling conditions, and the extraction method have been shown in previous publications (massire et al., ; shin et al., ) . after pcr amplification, the desalting method purifies the amplified nucleic acids, which are then loaded onto the esi-ms module. the mass of the resulting amplicon is determined and the nucleotide base composition of this amplicon is calculated based upon the mass and the known nucleotide sequences of the pcr primers and target genome regions. base compositions are determined for each unique amplicon in each well of the assay, and compared against a curated database. this database contains base composition signatures developed using data both gathered empirically and simulated from known sequences acquired from genbank. a final identification is then triangulated using the information generated from each assay primer pair. each sample processed on the pcr/esi-ms system included three controls introduced at various points in the process. ( ) an extraction control was added to every sample, which was amplified by a stand-alone primer pair included with the assay. failure to detect this control was indicative of an issue with sample extraction or other downstream failures. ( ) an amplification control, or calibrant, was part of the assay formulation for each well of the assay. each calibrant was a synthetic dna construct design to be amplified by its associated primer pair and to be easily distinguishable from organism targets by the analysis algorithm. the calibrant was used both to reduce low levels of background noise produced by the pcr and to control for amplification issues in the absence of target template. ( ) peptides of known masses were introduced during the desalting and esi-ms processes to provide a positive indication of the successful execution of the desalting and electrospray ionization steps, and to provide precise calibration points for the analysis of the spectra produced by the mass spectrometer. the analytical sensitivity of the assay was verified through low concentration testing of multiple strains of each fungus for which an lod was established. concentrations were determined by counting colony forming units (cfu) per milliliter. a "core" set of fungi was selected that exercised all assay primers and were part of the lod study. the core fungi were: aspergillus fumigatus, candida dubliniensis, candida glabrata, cryptococcus neoformans, and a mucor sp. an initial lod study was conducted using the five core fungi, utilizing sterile saline as a surrogate for clinical bal matrix. limits of detection were determined by processing -fold dilution series of five replicates each, and then confirming the lowest concentration at which all five replicates were positive for the target fungi by processing an additional replicates. the confirmed lod was the lowest concentration at which at least of replicates were positive for the target fungi. the limits of detection of additional fungi that tested the phylogenetic range of the assay were also established using this method described above. analytical specificity studies were conducted to evaluate the ability of the assay to perform in the presence of interfering substances and the ability of the assay to detect and characterize multiple target organisms in a single sample. a cross-reactivity study was conducted in which multiple species of bacteria, viruses and non-target fungi at high concentrations was tested. the assay reproducibility was evaluated by testing multiple low concentration replicates of the core fungi across multiple assay lots and systems. a total of bal samples were prospectively collected from consented patients in the bronchoscopy suite at the johns hopkins hospital, baltimore, md, usa, from july to december . inclusion: patients who had a clinical indication for bronchoscopy where bal was being sent for microbiologic testing; exclusion: patients who had tachycardia or hypotension at the time of bronchoscopy, or had a history of pulmonary hemorrhage syndrome, vasculitis, acute coronary syndrome, pregnancy, or were mechanically ventilated. written informed consent was obtained for patients who were interested in participating. participating subjects had an additional bal wash obtained for research purposes. standard of care laboratory testing data and clinical data were abstracted from chart review and de-identified prior to analysis of pcr/esi-ms. the study was approved by the johns hopkins institutional board review (irb# ). all bal samples were processed at the johns hopkins hospital microbiology laboratory for the detection and identification of fungal pathogens using all standard of care reference tests including direct microscopic examination by calcofluor white staining, fungal culture, galactomannan (positive cutoff value: gmi . ), and direct fluorescent antibody (dfa) microscopic examination that is the only method used for the detection of pneumocystis jirovecii. in parallel to the above reference standard of care tests, ml of direct bal fluid was tested by pcr-esi/ms (iridica™, ibis biosciences, ca, usa). patient chart review was conducted by two independent researchers. data was abstracted using a standardized data collection instrument and included sex, age, underlying conditions, histopathological findings, radiological results, antimicrobial regimen, clinical outcomes, and laboratory findings (galactomannan results in serum and bal, serum beta-d-glucan, and microscopy/culture results from tissues in addition to bals). patients with proven, probable, and possible fungal infections were defined by eortc/msg (european organization for research and treatment of cancer/invasive fungal infections and the national institute of allergy and infectious diseases mycoses study group) criteria (de pauw et al., ) , with some modification for p. jirovecii infection. probable pneumocystis infection would apply for these patients with immunocompromised conditions, clinical features, and radiological evidence but negative by microscopic examination. possible pneumocystis infection would apply for those patients with immunocompromised conditions and clinical features but no radiological and other laboratory findings. the limit of detection for the five core fungal species as well as an additional species were established (supplement fig. ). the lod of these fungal species ranged from b cfu/ml to cfu/ml with a mean lod of . cfu/ml and a median lod of . cfu/ml. additional strains of each species for which an lod was established were tested at near lod concentrations. the fungal species included in the lod and analytical sensitivity testing exercised the phylogenetic range and depth of the assay. the analytical specificity of the assay was evaluated by challenging the assay with high concentrations ( cfu/ml, copies/ml or tcid /ml) of non-target microorganisms: the assay and system were also evaluated with regards to their susceptibility to interference from endogenous and exogenous substances expected to be present within bal specimens. all substances, listed in supplement table were tested in the presence of near lod levels of the core fungi. no interference or spurious detections were observed. to evaluate the ability of the assay to correctly detect and identify target fungi in the presence of multiple targets, each of the core fungi was combined with one other member of the core set at near ( x) lod concentrations in triplicate. all components of each mixture combination were detected and no spurious detections were observed. additionally, a % detection rate was observed in reproducibility studies conducted using replicates of the core fungi at near lod concentrations, tested using multiple systems and assay lots. a total of patients were enrolled in this study and nonduplicated bal fluid specimens were collected over a two-year study period. two hundred and sixty-two of these samples were included in this study. the bals were excluded due to the following reasons: were due to insufficient sample volume (b ml); were due to invalid test results in pcr/esi-ms analysis; samples were not labeled. ninety-two percent of tested samples ( / ) were from patients known to be in an immunocompromised state. there were specimens from patients with transplantation ( . %, / ), from patients with malignancy ( . %, / ). the detailed underlying disease spectrum of these patients are listed in the supplementary tables - . twenty-three samples were obtained from immunocompetent patients. eighteen of these samples were negative by both reference standard assays and pcr/esi-ms. three samples were positive for yeast or candida albicans by both methods but none of these patients had clinical signs of fungal infection, most likely suggestive of colonization. one specimen was culture positive for histoplasmosis capsulatum but did not yield a detection by pcr/esi-ms. one specimen was detected by pcr/esi-ms as coccidioides immitis but found to be culture negative. the positive detection rate for reference methods was . % ( / ), and for pcr/esi-ms was . % ( / ) (table ) . we also assessed concordance on the genus and species levels for the results which were positive by both methods, finding a genus level concordance of . % ( / ) and a species level concordance of . % ( / ). the overall sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and positive likelihood ratio + (lr+) for broad level of fungal detection by pcr/esi-ms were . %, . %, . %, . %, and . , respectively (table ) . we then performed a structured chart-review of the patients which included clinical presentations, underlying conditions, imaging studies, and other laboratory markers. we found that specimens were from patients with probable or possible fungal infections in line with the factors illustrated in eortc/msg criteria (de pauw et al., ) . seventy-two of these specimens were from patients with transplantation ( . %, / ); from patients with malignancy ( . %, / ), and the remaining specimens were from patients with hiv, interstitial lung disease, chronic obstructive pulmonary disease, et al ( . %, / ) (fig. ) . the most common comorbidity was lung transplantation followed by hematological malignancy (fig. ) . detailed abstracted clinical data from the patient is listed in supplement table - . in the specimens from patients with a clinical context relevant to fungal infections, the sensitivity, specificity, ppv, npv and positive lr of pcr/esi-ms were . %, . %, . %, . %, and . , respectively (table ) . aspergillus was detected in bal samples by culture and/or pcr/ esi-ms, of which were considered to be related to lung infection by clinical context including underlying conditions, clinical features, and lung ct findings (fig. ) . eighteen of them were detected by pcr/ esi-ms; of which fifteen were verified by culture, and of which ten matched culture-based identification at the species level. thus, samples positive by both pcr/esi-ms and reference methods accounted for the majority of aspergillus infection-related cases ( %, / ). increased galactomannan values were detected in nine of these aspergillus infection-related specimens (ranging from . to . ), all of which were positive by both pcr/esi-ms and culture-based methods. of the clinically relevant aspergillus detections, none of the patients who were culture-positive had been given an antifungal agent before bal specimens were collected, while the patients who were positive by pcr/esi-ms but negative by culture had been given an azole before the specimen was taken. as to the two cases of aspergillus infectionrelated that were positive by culture but negative by pcr/esi-ms, one case presented with a positive galactomannan index (gmi . ). this patient suffered from lung cancer and underwent chemotherapy and radiotherapy. imaging showed tiny foci of cavitation within the anterior aspect of consolidation and some multifocal patchy foci of ground glass in both lungs, particularly in the middle and lower lung zones. the other case was a patient with idiopathic pulmonary fibrosis and left lung transplantation who was on intermittent voriconazole for months without any improvement. the culture grew aspergillus fumigatus and was positive for galactomannan (gmi . ). this patient had a mild fever with other serious respiratory symptoms and worsening lung function for several weeks. there were p. jirovecii detections by pcr/esi-ms, of which were concordant with the reference dfa results (table ) . thirteen patients were considered to have probable pneumocystis pneumonia according to their clinical presentations, which included fever, cough, worsening pulmonary function, and imaging alterations such as ground-glass lung infiltrates. the underlying conditions of the patients who had positive detection of p. jirovecii by pcr/esi-ms included hiv, hematological malignancy and liver transplant. the patients also positive by dfa were hiv cases. fourteen bals were detected positive for malassezia sp. by pcr/ esi-ms but none by culture. clinical evidence of possible fungal infection was found in two of these cases. one case was a years old male with lung transplantation and imaging showing patchy ground glass. the other case was a years old male with kidney transplantation and imaging showed bilateral consolidation with pleural effusions. analytical studies of the updated pcr/esi-ms platform demonstrated that the system and assay can detect a diverse array of fungal pathogens responsible for invasive fungal infections at low concentrations in a sterile saline matrix. one limitation was that the lod results generated in sterile saline matrix may not be the same as if they were done in clinical bal matrix. the system and assay were shown to provide reproducible, robust results in the presence of substances expected to be encountered in bal specimens, and did not generate unexpected detections or misidentifications in the presence of common bacterial and viral respiratory pathogens as well as these fungi not targeted in the assay, or when presented with specimens containing mixtures of low concentrations of fungi identified by the assay. to investigate the clinical performance of the system and assay, we evaluated bal specimens from patients for pulmonary fungal infection using pcr/esi-ms, and compared findings with stand of care reference tests. opportunistic fungal infections involve ubiquitous fungi and occur predominantly in individuals whose immune systems are compromised. in this study, % of the subjects were considered immune-deficient due to solid organ transplantation, bone marrow transplantation, neutropenia, chemotherapy and/or radiotherapy, aids, other chronic debilitating diseases, long-term usage of corticosteroids and/or use of immunosuppressants or cytokine antagonist medications. practically, it is essential to evaluate the diagnostic accuracy of microbiological methods on the basis of clinical relevance. a structured chart review was conducted for the specimens, of which specimens were from patients with probable and possible fungal infections based on the revised eortc/msg criteria (de pauw et al., ) . we found that the sensitivity, specificity, ppv, npv, and positive likelihood ratio of pcr/esi-ms were significantly increased when compared to reference laboratory methods alone. all of the patients were in an immunocompromised state, including transplant, hematological malignancy and aids patients who were the most vulnerable population at a high risk of invasive fungal diseases. although no fungal infections were found among those patients without predisposing immunocompromising factors, two fungal pathogens were detected in two patients. one strain of histoplasma capsulatum was isolated by culture but not detected by pcr-esi/ms (hence false negative) from a patient with multiple sclerosis and anemia, and imaging results showed a consolidation pattern and a . cm nodule in the right upper lobe, compatible with chronic histoplasmosis. one strain of coccidioides immitis was detected by pcr/esi-ms in a culture negative bal. the result was indeterminate since the -year-old male patient did present with cough, and the chest x-ray showed some filtrates as well as mediastinal adenopathy but lacked other lab results to support coccidioidomycosis infection and a history of travel to endemic areas was not available. he was diagnosed as unspecified ocular disease, and this pathogen could have been the causative agent for this patient's eye problems (vasconcelos-santos et al., ) . aspergillus sp. represent some of the leading causative agents of opportunistic fungal lung infection among immunocompromised patients (baughman, ; cadena et al., ) . aspergillus sp. was found to be a frequent colonizing pathogen in the airway in these populations. the development of invasive pulmonary aspergillosis depends on the level of the host immune deficiency . approximately % ( / ) of the isolated aspergillus cases were judged to be related to pulmonary infections. in the cases of probable pulmonary aspergillosis detected by pcr/esi/ms, % ( / ) were concordant with culture, and half of these cases had increased galactomannan values. the pcr/esi-ms system is rapid ( - hours) in comparison with culturebased methods, and can be performed directly on uncultured bal specimens, enabling earlier targeted therapy. in this study, there was no administration of antifungals before bronchoscopy for clinically relevant specimens which were culture-positive for aspergillus, while voriconazole, posaconazole, or amphotericin b had been used for those aspergillus detections by pcr/esi-ms, indicating the potential advantage of this approach in cases where prophylactic antifungal treatment has been initiated prior to specimen acquisition. although pcr methods potentially have high sensitivity, there were two probable aspergillosis cases missed by pcr/esi-ms, which could be due to poor quality of the specimens resulting in low yield of aspergillus dna (alanio and bretagne, ) . p. jirovecii remains one of the most common opportunistic pathogens in aids patients and occurs in patients with other causes of immunodeficiency (thomas jr. and limper, ) . the standard reference method for diagnosing p. jirovecii is a direct fluorescence microscopy assay on specimens obtained by bal (procop et al., ; choe et al., ) . there were additional p. jirovecii detections by pcr/esi-ms that were negative by dfa. we didn't confirm these cases by another molecular assay. however, all these patients were immunocompromised patients (table ) and all except one had clinical presentations and radiological evidence to support the clinical diagnosis of the disease. aids patients often have a high-burden of p. jirovecii, with a previous study showing dfa assay positive rate as high as to % (choe et al., ) . the detection rate by dfa in this study in the nine clinicalsuspicious-pcp-infection hiv patients was . % ( / ), while by pcr/ esi-ms was % ( / ). in the five non-hiv patients, detection of pneumocystis was negative by dfa but positive by pcr/esi-ms (table ) . patients whose immunocompromised state is not due to aids usually present a low burden of p. jirovecii and are difficult to diagnose microscopically (seah et al., ; fauchier et al., ) . our results showed positive p. jirovecii detection by pcr/esr-ms only in non-aids immunocompromised patients. this finding was consistent with previous studies demonstrating that real-time pcr assays increased sensitivity to detect p. jirovecii infection (hauser et al., ) . therefore, pcr/esi-ms could serve as a more rapid, sensitive and specific technology for the detection of p. jirovecii, especially in non-aids immunocompromised patients. certainly, more cases are needed for conclusive assessment. one case detected by pcr/esi-ms was thought to be free of pcp pulmonary infection due to lack of corresponding clinical presentations and radiological evidence for the infection. p. jirovecii can be colonized in both hiv and non-hiv immunocompromised patients (frealle et al., ) . although there are no definitive evidence suggesting the risk of reactivation and evolution of the colonization, the progression to pneumonia may need to be evaluated in cases of long-time usage of specific drugs such as cytokine blockers (morris and norris, ) . the fungal biomarker β-d-glucan could be an additional useful test with high negative predictive value for the diagnosis of the disease and prognostic value for monitoring the patient treatment (onishi et al., ; damiani et al., ; kutty et al., ) . malassezia sp. usually colonize the skin and occasionally colonize the respiratory tract. growth of malassezia requires specific fungal culture media, which is most likely to be the reason for the universally negative culture results for the organisms detected by pcr/esi-ms. recently, malassezia fungemia, peritonitis, arthritis and pulmonary infection have been reported in immunocompromised patients including neonates, diabetics with dialysis, and patients recovering from stem cell or solid organ transplantation (tragiannidis et al., ; velegraki et al., ; pedrosa et al., ) . patients on total parenteral nutrition are thought to at risk of this infection (baker et al., ) . although the two patients yielded clinically relevant data to support possible malassezia infection detected by pcr/esi-ms in this study, the definitive diagnosis of malassezia infection in these cases needs to be confirmed by another molecular method. both patients were in a severely immunocompromised state, and lived only short periods of time following collection of the specimens studied here. pcr/esi-ms offered a way to detect this potential pathogen in an immunocompromised population. the overall sensitivity and specificity of the pcr-esi/ms was found to be . % and . % (respectively), with a positive likelihood ratio of . in those patients who were categorized as having proven, probable or possible invasive fungal infection per the eortc-msg guidelines. this demonstrates the clinical utility of the technology, especially when applied judiciously within the appropriate treatment context. additionally, in a clinical setting these results would be provided to the treating clinician within hours, as opposed to the days to weeks that can be required for the results generated from culture based methods. the range of fungi which can be detected by the technology also reduces the testing burden required for a given patient, as current molecular technologies can generally only detect a limited number of fungi. furthermore, the automated molecular identification provided by the system does not require specialized training and knowledge required for microscopic identification of fungi from culture or histopathological specimens. it has been noted that improving the ease and rapidity of fungal diagnosis will have a positive effect on patient outcomes and hospital costs (arvanitis et al., ) . pcr-esi/ms is not the only broad spectrum molecular technology that is under development for the diagnosis of invasive fungal infections. multiplex pcr and dna sequencing analysis of patient specimens are among the technologies that can provide a utility similar to that which has been demonstrated here for pcr-esi/ms. the broadening of the diagnostic toolkit available to clinicians, and the accompanying gains in speed and accuracy of diagnosis, could help to improve patient outcomes, reduce hospital costs and help combat the spread of antibiotic resistance. unfortunately, pcr-esi/ms was removed from the market by abbott laboratories in primarily due to financial consideration (ozenci et al., ) . however, our present data, as well as a recent publication using pcr-esi/ms to improve fungal diagnosis in hematological patients (krifors et al., ) , sheds new light on the importance of developing these non-culture based broad panel molecular assays to enhance the detection of fungal pathogens in those patients with invasive fungal infections. the continuing development and clinical investigation of these technologies should be pursued. difficulties with molecular diagnostic tests for mould and yeast infections: where do we stand? update from the laboratory: clinical identification and susceptibility testing of fungi and trends in antifungal resistance molecular and nonmolecular diagnostic methods for invasive fungal infections malassezia pneumonia: a rare complication of parenteral nutrition therapy infectious complications part respiration; international review of thoracic diseases invasive aspergillosis: current strategies for diagnosis and management diagnostic value of direct fluorescence antibody staining for detecting pneumocystis jirovecii in expectorated sputum from patients with hiv infection combined quantification of pulmonary pneumocystis jirovecii dna and serum ( -n )-beta-d-glucan for differential diagnosis of pneumocystis pneumonia and pneumocystis colonization revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients diffusion of pneumocystis jirovecii in the surrounding air of patients with pneumocystis colonization: frequency and putative risk factors prevalence of immunosuppression among us adults prospective clinical evaluation of respiratory samples from subjects at risk for pneumocystis jirovecii infection by use of a commercial real-time pcr assay pcr with electrospray ionization-mass spectrometry on bronchoalveolar lavage for detection of invasive mold infections in hematological patients ja ( ) beta-glucans are masked but contribute to pulmonary inflammation during pneumocystis pneumonia pcr followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens colonization by pneumocystis jirovecii and its role in disease diagnostic accuracy of serum , -beta-d-glucan for pneumocystis jirovecii pneumonia, invasive candidiasis, and invasive aspergillosis: systematic review and meta-analysis demise of polymerase chain reaction/electrospray ionization-mass spectrometry as an infectious diseases diagnostic tool executive summary: practice guidelines for the diagnosis and management of aspergillosis: update by the infectious diseases society of america malassezia infections with systemic involvement: figures and facts culture-independent molecular methods for detection of antifungal resistance mechanisms and fungal identification the global problem of antifungal resistance: prevalence, mechanisms, and management detection of pneumocystis jirovecii in respiratory specimens by four staining methods comparison of the fxg: resp (asp+) real-time pcr assay with direct immunofluorescence and calcofluor white staining for the detection of pneumocystis jirovecii in respiratory specimens detection, identification, and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus pcr coupled with electrospray ionization/mass spectrometry pneumocystis pneumonia minireview: malassezia infections in immunocompromised patients chronic coccidioidomycosis endophthalmitis without concomitant systemic involvement: a clinicopathological case report malassezia infections in humans and animals: pathophysiology, detection. and treatment clinical performance of aspergillus pcr for testing serum and plasma: a study by the european aspergillus pcr initiative enhancing molecular approaches for diagnosis of fungal infections this work was funded in part by a contract from abbott, inc. with johns hopkins university school of medicine. dr. rothman was also supported in part by nih niaid hhsn c.we would like to thank dr. rex yung and the interventional pulmonology team as well as all other pulmonologist at the johns hopkins hospital for their contribution in helping with collecting the bals for the study. supplementary data to this article can be found online at https://doi. org/ . /j.diagmicrobio. . . key: cord- -g m y x authors: sacco, oliviero title: il lavaggio broncoalveolare (bal) in età pediatrica date: journal: pneumologia interventistica doi: . / - - - - _ sha: doc_id: cord_uid: g m y x il lavaggio broncoalveolare o bal, permettendo di ottenere le cellule ed i soluti presenti sulla superficie epiteliale del tratto respiratorio distale, si è dimostrato una metodica di ricerca essenziale per lo studio dei meccanismi eziopatogenetici delle malattie del polmone profondo, come ad esempio lo studio delle interstiziopatie, su cui esiste una vastissima letteratura di dati ottenuti con il bal. oltre a questo aspetto di metodica di ricerca, il bal rappresenta perè anche una procedura diagnostica insostituibile nella pratica clinica quotidiana. eventuale riflesso vagale non è così diffusamente impiegato in campo pediatrico come nell'adulto. essendo un farmaco di premedicazione, la sua somministrazione sc almeno - minuti prima della broncoscopia, come spesso si usa negli adulti, generalmente causa il pianto del bambino, lo agita e non ne favorisce il suo arrivo in sala operatoria rilassato e sereno, per cui molto spesso si evita il suo impiego. se usata, se ne consiglia la somministrazione ev lenta ( - mg/kg); qualora il paziente presenti un frequenza cardiaca superiore ai / , il suo uso deve essere ulteriormente ponderato [ ] . il bal viene generalmente eseguito con l'impiego di un fibro-o videobroncoscopio pediatrico con canale operatore, il cui diametro esterno è generalmente di , mm nei bambini inferiori a - anni (peso corporeo [ ] [ ] [ ] [ ] [ ] [ ] kg) e con strumenti con diametro esterno di , - , mm nei pazienti più grandi. la sede di esecuzione del bal è generalmente nel bronco segmentario che afferisce alla zona di parenchima sede della patologia in studio se il processo è focalizzato mentre, nel caso di patologie generalizzate, la sede del bal è preferenzialmente nel lobo medio o nella lingua. questi distretti, speculari tra loro, sono le sedi preferite per l'esecuzione della metodica perché, quando il paziente giace supino, la loro posizione è tale da favorire il recupero del liquido instillato. non diversamente che nell'adulto il broncoscopio deve essere fatto procedere nel bronco prescelto fino a far sì che la punta dello strumento si incunei nel bronco, occludendone il lume ed impedendo così che il liquido instillato refluisca verso le vie aeree centrali invece di arrivare al parenchima. nei bambini di età inferiore agli - anni e con peso corporeo < - kg, qualora non si riesca ad incuneare bene il broncoscopio nel bronco lobare medio o lingulare per le loro piccole dimensioni, come seconda scelta elettiva si può eseguire il bal nella piramide basale del lobo inferiore destro o sinistro, di facile accesso. si ricorda che, nel caso in cui vi siano le indicazioni ad eseguire il bal in due segmenti polmonari, uno sede di malattia, uno di normale aspetto radiologico, è consigliabile eseguire la metodica prima nella zona di parenchima sano, così che il canale operativo dello strumento non venga contaminato, come avverrebbe invece se il bal fosse prima eseguito nella zona affetta. queste considerazioni sono particolarmente importanti nel caso di infiltrati polmonari di sospetta natura infettiva. nell'adulto la metodica più comunemente accettata per eseguire il bal è quella di instillare cinque aliquote di ml ciascuna di soluzione fisiologica sterile (preferibilmente a temperatura corporea). così facendo ed aspirando dopo ogni aliquota, la prima frazione recuperata è più rappresentativa della citologia bronchiale: più ricca di neutrofili e meno di linfociti rispetto alle aliquote seguenti, che a loro volta sono più rappresentative della citologia e delle varie componenti non-cellulari di origine alveolare. nell'adulto si tende quindi a raccogliere separatamente la prima aliquota instillata, mentre le seguenti verranno aspirate nello stesso contenitore [ ] . in età pediatrica la quantità di liquido instillato non è chiaramente fissa, ma dipende dal peso corporeo del paziente: da a ml di soluzione fisiologica/kg, scegliendo i volumi più piccoli quanto più le condizioni del paziente sono critiche. anche se non si è ancora raggiunta capitolo endoscopia bronchiale pediatrica un'universale standardizzazione della metodica, attualmente è sempre più diffusa l'abitudine di instillare tre aliquote uguali, ognuna del volume di ml/kg peso corporeo [ ] . si deve però anche considerare che, per ottenere un sufficiente recupero da ogni aliquota, il volume della stessa non dovrebbe comunque essere minore di ml, considerando che il recupero mediamente è del %- %. ne consegue che, fino ad un peso corporeo di - kg, non è generalmente fattibile raccogliere separatamente le diverse aliquote: instillando - ml di fisiologica in totale, se il recupero è di - ml, una suddivisione di questo in una prima aliquota bronchiale ed una seconda alveolare può divenire problematica proprio per l'esiguità del volume del liquido recuperato. la metodica di suzione impiega generalmente un aspiratore che genera una pressione negativa non troppo elevata ( - mmhg), in quanto le vie aeree dei bambini sono più facilmente collassabili rispetto a quelle degli adulti, l'aspiratore viene collegato ad una provetta che funge da trappola per il liquido recuperato. quando il paziente è in uti, intubato e ventilato, si usa un connettore a forma di "y" per permettere il proseguimento della ventilazione durante l'introduzione del broncoscopio nel tubo tracheale. un broncoscopio pediatrico standard, del diametro esterno di , - , mm, può passare attraverso in tubo tracheale col diametro interno di almeno , - , mm e permettere al tempo stesso una minima ventilazione. i tubi tracheali con diametro < , mm possono essere facilmente incannulati solo dai più piccoli broncoscopi neonatali con il diametro esterno di solo , mm, ma questi strumenti sono privi di canale operativo. in questi pazienti, intubati con un piccolo tubo tracheale, il bal può essere comunque eseguito usando un semplice catetere (o un catetere a palloncino, misura - ranch), che andrà spinto in periferia fino al suo incuneamento in un bronco subsegmentario, possibilmente facendolo passare attraverso un diaframma, così che possa essere mantenuta la pressione positiva della ventilazione [ ] . con tale metodica il punto di esecuzione del bal non può essere determinato con la stessa accuratezza che con il broncoscopio, ma almeno si riesce a guidarne l'introduzione nel bronco principale di scelta: piegando, infatti, la testa del paziente verso sinistra al momento dell'introduzione del catetere, lo stesso verrà indirizzato preferenzialmente verso il bronco principale di destra, e viceversa. con la sola eccezione della mancanza di accuratezza del punto di esecuzione della metodica (a parte la scelta del lato), il bal eseguito "alla cieca" con catetere fornisce dati equivalenti a quello eseguito con broncoscopio, almeno nei pazienti con problematiche polmonari diffuse, come le bronchioliti da vrs [ ] . l'impiego del bal nei pazienti ventilati è un esempio della sua grande utilità clinica per distinguere le vere polmoniti del paziente ventilato che non rispondono all'iniziale trattamento antibiotico, da tutte le altre condizioni che inducono infiltrati polmonari non su base infettiva: le emorragie polmonari, l'ards, le polmoniti eosinofiliche, l'edema su base cardiogena, la tossicità da farmaci, per non citarne che le principali [ ] . il paziente, sottoposto a bal nel corso della mattinata, può sviluppare una singola puntata iperpiretica alcune ore dopo, generalmente nel pomeriggio, a risoluzione spontanea ed eziologia in-certa. questa complicanza più banale e comune del bal ha una frequenza che, nella nostra casistica, avviene una volta ogni - procedure. solo molto raramente la febbre persiste e può essere considerata una complicanza vera, effettivamente correlata a diffusione di un processo infettivo. l'esecuzione del bal, prolungando di solo - minuti l'endoscopia, di per sé non aumenta che marginalmente il rischio di ipercapnia ed ipossia, a patto che il paziente venga ventilato correttamente e che il broncoscopio venga fatto passare attraverso il tubo a "t" mentre il paziente è ventilato in maschera, come sovraesposto. anche se naturalmente ogni tipo di alterazione della coagulazione può di per sé aumentare il rischio di sviluppare un'emorragia mucosale, queste sono molto rare, se si applica una gentile suzione nel recuperare il liquido instillato. il bal è stato eseguito anche in bambini con un'importante piastrinopenia ( plt/mm ), senza che si sviluppassero complicazioni emorragiche. queste in realtà, più che a livello del punto di esecuzione del bal, possono avvenire più frequentemente a livello della coana ove passa lo strumento [ ] . il procedimento per trattare e studiare il balf proveniente da un paziente pediatrico non è diverso da quello in uso negli adulti. la provetta di raccolta del balf deve essere subito raffreddata a °c in acqua/ghiaccio ed inviata al laboratorio appena possibile. se esiste una prima aliquota del balf raccolta separatamente, questa viene generalmente impiegata per studi colturali così come è stata ottenuta dal paziente, per non perdere carica batterica o alterarne la composizione. se è indicata (raramente) la ricerca di anaerobi, il liquido deve essere inoculato nell'apposito medium di coltura appena possibile. le restanti aliquote del balf vengono poi filtrate attraverso una garza sterile per allontanare il muco quindi, per lo studio della quota cellulare, il liquido viene centrifugato a - g per - minuti. il sovranatante viene quindi aspirato e generalmente conservato a - °c per eventuali ulteriori studi di proteomica, al momento più di interesse di ricerca che clinico [ ] . il pellet cellulare ottenuto con la centrifugazione è risospeso in , - ml di medium per coltura cellulare, e la sospensione cellulare così ottenuta viene usata per l'allestimento di citopreparati, ottenuti generalmente con l'impiego di una citocentrifuga. se questa non è disponibile, si possono allestire strisci su vetrini, non diversamente di quanto in uso in ematologia. in ogni caso è utile allestire diversi vetrini, anche se solo alcuni verranno usati al momento. le colorazioni più usate per la citologia sono il may-grunwald giemsa ed il diff-quick (merz & dade a.g., dudigen, germany); a quest'ultima colorazione va spesso la preferenza dei laboratori di pneumologia, in quanto tutto il ciclo di colorazione si completa in circa minuti. il limite del diff-quick, come riferito da diversi anatomopatologi, è quello di non dare la miglior definizione della cromatina nucleare, particolare importante per l'identificazione di eventuali cellule neoplastiche. l'indicazione alla ricerca di cellule tumorali, molto comune nell'età adulta, nei pazienti pediatrici è comunque per fortuna molto più rara. per eseguire la conta differenziale è necessario identificare almeno - cellule, quindi eseguire le percentuali. le eventuali cellule epiteliali devono concorrere a determinare la cellula-rità totale del balf, ma le stesse sono generalmente escluse dal calcolo delle percentuali della conta differenziale. qualora si ottenga ripetutamente un'alta contaminazione da cellule epiteliali (> %) nel balf, si incorre con tutta probabilità in errori di esecuzione della metodica, di cui ricordiamo i più comuni. ) la pressione di aspirazione è troppo elevata, con conseguente collasso delle pareti bronchiali (tanto più probabile fino ai - anni di età) e facilità di suzione della mucosa nel canale operativo dello strumento; il collabimento della parete bronchiale è comunque facilmente verificabile visivamente durante l'esecuzione della metodica. ) il broncoscopio non è bene incuneato nel bronco ed in asse con il lume bronchiale, ma la sua estremità punta contro le pareti bronchiali con conseguente difficoltà nel recupero del balf, in cui appariranno anche eritrociti oltre che cellule epiteliali. ) lo strumento non viene tenuto fermo durante la metodica, ma va avanti e indietro lungo il lume bronchiale, con effetto "spazzola". poiché non è etico eseguire il bal in bambini sani solo a scopo di studio, i valori di "normalità" sono stati ottenuti durante endoscopie eseguite per indicazioni cliniche che venivano ritenute non correlabili con una patologia bronco-polmonare quali laringomalacia, stenosi delle vie aeree centrali, follow-up di inalazione pregressa di corpi estranei etc., o al momento dell'esecuzione di anestesia generale per interventi chirurgici non associabili a patologia respiratoria. in questo caso il bal veniva eseguito per lo più senza l'uso di un broncoscopio, ma usando un catetere introdotto lungo il tubo tracheale ed incuneato alla cieca in un bronco subsegmentario. tutti i pazienti presentavano normale radiografia polmonare, normale funzionalità respiratoria, assenza di segni clinici o laboratoristici di infezioni sistemiche e/o a carico delle alte o basse vie aeree. con queste limitazioni di relativa "normalità", la cellularità totale/ml di balf è risultata variare di molto tra i diversi studi: da cellule/ml a /ml, con variazioni che anche risentono dell'età dei pazienti, in quanto pazienti più piccoli tendono ad avere cellularità totale più elevata, non diversamente dai gb del sangue periferico [ ] [ ] [ ] . si consiglia, nei pazienti pediatrici, di mantenere come limite superiore di normalità le cell/ml di balf, valore oltre il quale si può parlare effettivamente di aumento della cellularità totale o assoluta alveolare. queste considerazioni sui valori della cellularità totale sono chiaramente validi solo in presenza di un bal eseguito in maniera corretta: quanto più elevata la quota di cellule epiteliali contaminanti (sia squamose, di derivazione orofaringea, sia cigliate della mucosa bronchiale), quanto meno sono valide queste considerazioni sulla cellularità totale. se le cellule epiteliali sono > %- % di quelle recuperate, il bal non è stato eseguito correttamente, e non si può più parlare di valori normali di riferimento della cellularità totale (fig. ) . la conta differenziale delle cellule presenti nei balf ottenuti in età pediatrica può essere paragonata a quella riscontrata nei controlli adulti. facendo la media tra i diversi studi pubblicati e dalla nostra esperienza, i valori "normali" di riferimento usati nel nostro laboratorio, possibilmente scartando la prima aliquota del balf sono i seguenti: • macrofagi: %- % • linfociti: %- % • neutrofili: %- % • eosinofili: %- , % riguardo al giudizio di una buona esecuzione del bal, poiché la popolazione macrofagica è la preponderante, ed il macrofago è la vera cellula residente alveolare, solo la presenza di una il lavaggio broncoalveolare (bal) in età pediatrica discreta quota di macrofagi (almeno %- %) nelle aliquote di balf susseguenti la prima è la prova che il liquido di lavaggio ha effettivamente raggiunto il parenchima polmonare. la conta differenziale è essenziale per rispondere al quesito che è alla base dell'indicazione per l'esecuzione del bal: esiste alveolite, ovvero infiammazione del parenchima polmonare, e se la risposta è affermativa, di quale tipo? la distinzione fondamentale è tra: • alveolite neutrofilica: polmoniti batteriche, bronchite cronica, vari tipi di fibrosi/interstiziopatie polmonari, inalazione ricorrente di materiale gastrico da eventi di reflusso gastroesofageo, asma; • alveolite eosinofilica: polmoniti eosinofiliche, asma, aspergillosi allergica broncopolmonare, sindrome di churg-strauss (estremamente rara nei bambini), reazioni a farmaci; • alveolite linfocitaria: polmonite da ipersensibilità, tubercolosi, artrite reumatoide, polmonite interstiziale linfocitaria nell'aids, etc.). stabiliti i valori indicativi di normalità per neutrofili e linfociti, ogni aumento significativo rispetto a questi (indicativamente almeno il %) può deporre per la presenza di alveolite. in presenza di una vera alveolite, l'aumento di una quota cellulare dovrebbe poi causare anche l'aumento consequenziale della cellularità totale [ ] . qualora si sia in presenza di un'alveolite linfocitaria, per valutare quanto il processo sia attivo, oltre alla percentuale dei linfociti presenti, si può ricorrere alla determinazione delle sottopopolazioni o fenotipo linfocitario, identificando i markers di attivazione sulla superficie cellulare: rapporto tra cd e cd , espressione di molecole di classe ii hla dr, recettore per il- , etc. È consigliabile che questo esame sia eseguito in contemporanea sui linfociti del balf e su quelli ricavati dal sangue periferico: lo stato di attivazione linfocitaria d'organo (locale) può essere così paragonato allo stato di attivazione presente a livello ematico (sistemico). È razionale richiedere lo studio delle sottopopolazioni linfocitarie solo qualora si sia in presenza di una vera linfocitosi (linfociti > % nella conta differenziale). la prevalenza di cellule cd + si ha nella sarcoidosi e nel morbo di crohn, mentre prevalgono i linfociti cd + nelle polmoniti da ipersensibilità, nell'istiocitosi x, nelle polmoniti da farmaci, nella boop [ ] . . bal con intensa contaminazione di cellule cigliate della mucosa bronchiale in un caso in cui il recupero del liquido di lavaggio era stato scarso e l'aspirazione era perdurata più del dovuto per tentare di aumentare il recupero:bal non eseguito correttamente capitolo endoscopia bronchiale pediatrica gli anticorpi monoclonali si usano anche nella diagnosi dell'istiocitosi polmonare, ove si ricercano le cellule cd +: qualora la loro percentuale > %, la diagnosi è molto probabile. percentuali minori non sono invece ritenute diagnostiche, in quanto %- % di cellule cd + possono essere presenti anche in pazienti normali [ ] . il bal è la metodica essenziale nella diagnostica delle emorragie alveolari, pregresse o in atto (nel bambino essenzialmente nell'emosiderosi polmonare idiopatica e nell'alveolite emorragica da chemioterapia oncologica, più raramente nelle vasculiti associate alle malattie del collageno). la diagnosi infatti si basa sulla ricerca del ferro nel citoplasma dei macrofagi alveolari che, con la colorazione di mallory diviene evidente: si visualizzano i siderofagi, macrofagi che hanno fagocitato eritrociti giunti nell'alveolo. se l'emorragia alveolare risale a settimane prima del bal, si potranno apprezzare solo i depositi di emosiderina, se è un fatto recente (giorni), i gr fagocitati saranno ancora visibili all'interno dei macrofagi, in varie fasi di degradazione. se l'emorragia è in atto, la prima aliquota del lavaggio (bronchiale) può non presentare colorazione ematica, che invece diventerà sempre più evidente nelle aliquote successive (alveolari), che assumeranno via via un aspetto più francamente ematico. qualora si sospetti un'alveolite emorragica vi è pertanto la massima indicazione alla raccolta frazionata non solo della prima aliquota del balf ma anche di ogni aliquota successiva [ ] . si ricorda qui incidentalmente che, il riscontro di gr nel balf senza eritrofagocitosi in atto e senza una significativa quota di siderofagi è da considerare solo come contaminazione, avvenuta durante l'aspirazione del balf. analogamente alla colorazione di mallory che rende evidenti gli accumuli di ferro nei macrofagi alveolari, così le colorazioni con il nile red o con il red oil visualizzano i vacuoli di grasso nel citoplasma dei macrofagi, che vengono così detti lipofagi (fig. ) . qualche raro lipofago si può riscontrare anche nel balf di un soggetto normale, ma il loro numero può aumenta- fig. . colorazioni con nile red di macrofagi alveolari,che presentano positività di vario grado come contenuto intracitoplasmatico di gocciole di grasso,così che il loro score varia da (negatività al nile red) a score (totale scomparsa di ogni dettaglio cellulare).i macrofagi alveolari positivi spiccano per presenza di gocciole al loro interno,di un colore giallo brillante il lavaggio broncoalveolare (bal) in età pediatrica re considerevolmente in patologie molto diverse, che inducono tutte l'accumulo nel loro citoplasma di grassi di origine endogena o esogena. l'accumulo di lipidi di origine endogena può avvenire nelle bronchiectasie, fibrosi polmonari, polmoniti ostruttive, tumori polmonari: tutte condizioni che causano turbe nel metabolismo del surfactante, i cui prodotti di degradazione si possono così accumulare all'interno del citoplasma macrofagico. nell'età pediatrica la condizione clinica che più frequentemente si associa ad un aumento dei lipofagi non è una di queste, bensì l'arrivo nell'alveolo di grassi esogeni, con l'inalazione cronica di materiale alimentare. i macrofagi, che fagocitano le sostanze estranee giunte nell'alveolo, il cui accumulo potrebbe interferire con la funzionalità della membrana alveolo-capillare, nel caso dell'inalazione di materiale alimentare riescono a catabolizzare proteine e glucidi ma non i grassi, che così si accumulano con il tempo nel loro citoplasma. questo può avvenire: ) per turbe della deglutizione, soprattutto nei pazienti con seri problemi neurologici e conseguente incoordinazione motoria, oppure ) nella malattia da reflusso gastro-esofageo. in quest'ultimo caso il materiale gastrico può risalire l'esofago fino a passare lo sfintere esofageo superiore e bagnare il laringe e, qualora i riflessi tussigeni non siano sufficientemente validi, causare inalazione di materiale alimentare di origine gastrica. queste inalazioni, molto piccole di volume ma ricorrenti, non causano le classiche polmoniti da inalazione massiva di cibo, ma inducono al massimo la comparsa di sfumati infiltrati parenchimali nelle parti declivi dei polmoni [ ] . per quantificare la presenza di lipofagi nel balf, si ricorre ad una valutazione semi-quantitativa degli stessi, consistente nel valutare in maniera sequenziale macrofagi e dando a ciascuno di essi uno score tra (assenza totale di ogni gocciola di grasso nel citoplasma) e (gocciole di grasso così numerose e grosse da oscurare anche il nucleo e gli altri dettagli cellulari). sommando lo score di macrofagi si arriva così a formulare il cd lipid index (li), il cui valore teorico può variare da (assenza totale di lipofagi) a (tutti i lipofagi presentano score ). poiché rari lipofagi alveolari si possono riscontrare anche nel balf di soggetti normali, esiste il problema oggettivo di fissare un valore cut-off per il li, oltre il quale si passa dalla normalità alla patologia. in letteratura tale valore è stato fissato generalmente a [ ] , ma tali studi sono stati fatti includendo anche gran parte di pazienti neurologici, con inalazione cronica di cibo durante la deglutizione. in questi pazienti le turbe della deglutizione si possono sospettare già semplicemente verificando se compare tosse durante il pasto, o con ulteriore sicurezza con lo studio della deglutizione con mdc, che se inalato genera una tracheo/broncografia. in realtà in questi pazienti non vi sono quindi le indicazione ad eseguire un bal per ricerca dei lipofagi. invece nei pazienti con sintomi respiratori ricorrenti (laringo e/o broncospasmi, tosse, infezioni broncopolmonari) di incerta origine e poco responsivi alle usuali terapie mediche, in cui si possa sospettare che in realtà alla base vi possa essere una misconosciuta malattia da reflusso gastro-esofageo con sintomi sovraesofagei, vi sono forti indicazioni ad eseguire un bal, proprio per la ricerca dei lipofagi alveolari. sottoponendo questi pazienti, senza problemi neurologici e senza inalazione alla deglutizione, sia alla phmetria esofagea che al bal, nella nostra esperienza un valore di lipid index > era significativamente correlato con una phmetria positiva per rge. una phmetria patologica non si associa però invariabilmente ad un li> . in pazienti con una phmetria positiva per rge e sintomi respiratori, non sempre questi derivano dall'inalazione di materiale alimentare nelle via respiratorie: basta che si instauri un'esofagite per causare tosse riflessa, ed anche quando il reflusso è tale da risalire fino al laringe, il laringospasmo o la tosse che scatena vanno intensi come meccanismi di difesa, tendenti a proteggere le via aeree inferiori dall'inalazione. nella nostra esperienza è più facile trovare valori patologici di lipid index entro capitolo endoscopia bronchiale pediatrica i - anni di età piuttosto che nei pazienti più grandi, quando i riflessi di difesa come la tosse diventano più validi [ ] . È poi importane notare che valori patologici di li sono facilmente associati ad uno spiccato aumento della cellularità totale, dovuto esclusivamente ad uno spiccato aumento della quota di neutrofili, che può arrivare fino al %- % della conta differenziale (fig. ) . tale aumento, paragonabile a quello che si riscontra nei balf ottenuti nelle polmoniti batteriche, si accompagna ad esami colturali negativi per germi comuni. fig. . bal con assoluta predominanza di neutrofili in un caso di un bambino di anni con malattia da reflusso gastroesofageo e sintomi respiratori ricorrenti: broncospasmo ricorrente/persistente, tosse scatenante vomito. l'estrema neutrofilia del bal (non dissimile da quella che si può riscontrare in un episodio di broncopolmonite batterica) può rende difficile l'esecuzione e la lettura del lipid index abbiamo potuto dimostrare che l'afflusso di neutrofili è causato dalla produzione da parte della mucosa bronchiale di il- , chemochina che induce la chemiotassi dai neutrofili verso la sua zona di rilascio. la secrezione di questa interleuchina è la risposta della mucosa bronchiale all'esposizione, durante le ricorrenti microinalazioni, al ph acido dei succhi gastrici, tanto che un efficace tamponamento gastrico come quello ottenuto con gli inibitori della pompa protonica (ppi) tende a normalizzare la percentuale dei neutrofili [ ] . in conclusione, la ricerca dei lipofagi alveolari e la loro quantizzazione con il li nei pazienti con sospetto clinico di reflusso gastro-esofageo è l'unica maniera per dimostrare che effettivamente gli eventi di reflusso sono tali da causare ricorrenti microinalazioni. la forza di questa indagine risiede anche nel fatto che, essendo il macrofago la cellula residente a livello alveolare, con un'emivita di alcuni mesi, il li è la somma temporale delle inalazioni che hanno raggiunto il polmone profondo nei mesi precedenti, non diversamente dall'hb glicosilata, espressione dei valori medi della glicemia nelle settimane precedenti al prelievo. nella proteinosi alveolare, sia congenita che acquisita, il balf ha un aspetto lattescente. nei preparati con citocentrifuga e colorati con may-grunwald giemsa si osservano voluminosi macrofagi alveolari, il cui citoplasma è ripieno di vacuoli, intervallati da materiale amorfo extracellulare basofilo. questo materiale e le inclusioni citoplasmatiche dei macrofagi mostrano una colorazione rosa con l'acido periodico di schiff. la caratterizzazione poi delle componenti del surfactante che si accumulano nel bal, come le apoproteine del surfactante, permette poi una miglior caratterizzazione del tipo di proteinosi, con differenziazione delle forme primitive o congenite da quelle secondarie o acquisite [ , ] . il balf è molto utile per la ricerca di microrganismi, ma l'interpretazione delle comuni indagini microbiologiche eseguite su di esso è complicata dalla frequente contaminazione da parte di materiale orofaringeo che il paziente può inalare durante l'endoscopia, quando le corde vocali sono tenute aperte dal passaggio dell'endoscopio. considerazioni simili devono essere tenute in mente quando si passa con l'endoscopio attraverso il rinofaringe: se vi sono delle secrezioni che oscurano la visuale è saggio non rimuoverle aspirandole attraverso l'endoscopio, se non si vuole che il canale operativo sia contaminato dalla flora (mista) del canale orale. questo è tanto più importante qualora si debbano studiare gli infiltrati polmonari dei pazienti immunocompromessi: in questi casi anche germi classificati come saprofiti ed usuali commensali del cavo orale possono invece essere i veri responsabili della patologia polmonare [ ] . i criteri su cui ci si basa per distinguere le vere infezioni polmonari dalla semplice contaminazione sono essenzialmente: ) esami colturali con valutazione semiquantitativa della carica batterica. lo sviluppo di più di di colonie batteriche, inoculando nel terreno di coltura ml di balf della quota alveolare, si è dimostrato diagnostico per la presenza di broncopolmonite. i dati microbiologici devono però essere sempre correlati con gli altri dati clinici: anche lo sviluppo di specie microbiche saprofite del cavo orale può avere importanza clinica se il paziente è immunocompromesso. ) ricerca diretta dei batteri. la colorazione standard è quella di gram; mentre la presenza di cellule epiteliali squamose con batteri adesi alla superficie è prova di inalazione di materiale orale, il riscontro di neutrofili e/o macrofagi fagocitanti batteri è un sicuro indice di infezione polmonare in atto (fig. ) . per la ricerca diretta di altri patogeni si ricorre a colorazioni specifiche o a metodi- fig. . paziente con distelettasia del lobo medio ed infezioni ricorrenti in tale sede; bal eseguito nel lobo medio in periodo di benessere. la discreta quota di neutrofili è spia delle infezioni ricorrenti,anche se l'esame colturale del bal è poi risultato negativo capitolo endoscopia bronchiale pediatrica che di biologia molecolare. con la colorazione argentina di grocott si evidenziano bene sia le ife fungine che le inclusioni intracellulari caratteristiche del pneumocystis carinii. con la colorazione di ziehl-neelsen si identificano classicamente i micobatteri. per la diagnostica precoce delle infezioni virali come nel caso del citomegalovirus si impiegano tecniche di immunoistochimica e di immunofluorescenza, che impiegano anticorpi monoclinali, specifici per i vari antigeni virali. ) tecniche di biologia molecolare. si ricerca una particolare sequenza di basi (dna o rna), specifica di un determinato patogeno. potenti metodiche di amplificazione del segnale, come la polymerase chain reaction (pcr), permettono di moltiplicare anche per molti ordini di grandezza la sequenza genica in studio (in teoria basta la presenza anche di una sola copia della sequenza in studio). la pcr attualmente è molto impiegata in clinica nella ricerca di cariche batteriche molto esigue, come spesso avviene nelle micobatteriosi. in questo particolare campo di applicazione la pcr permette inoltre la conferma di infezione entro poche ore, senza dover aspettare il risultato di lunghi esami colturali, come è la regola nelle micobatteriosi [ ] . la biologia molecolare assume anche un importante ruolo nella dimostrazione di eventuali multiresistenze ai farmaci da parte del micobatterio tubercolare, in quanto lo sviluppo della multiresistenza si associa a specifici marcatori genici [ ] . riguardo a tutti questi studi di microbiologia, per una corretta valutazione dei risultati, bisogna comunque sempre tener presenti queste considerazioni: • la presenza di un microrganismo nel balf è diagnostica solo se lo stesso non è mai anche un semplice colonizzatore delle vie aeree, come può accadere invece nel caso dell'aspergillo, candida, micobatteri atipici, cmv, hsv. l'identificazione invece di un organismo che abitualmente non si trova nelle vie aeree, come il pneumocistis carinii (fig. ) , m. tuberculosis, mycoplasma pneumoniae, legionella, nocardia, histoplasma, blastomyces, virus influenzali, rsv, è di per sé diagnostica. • per le considerazioni sovraesposte, le tecniche di pcr, amplificando enormemente il segnale, sono quelle che in particolare possono generare falsi positivi, identificando organismi che non sono cause di infezione. per evitare questo, attualmente si usano tecniche di pcr semiquantitative [ ] . • ogni risultato deve sempre essere valutato nel contesto clinico generale, con particolare riguardo allo stato immunitario del paziente nonché, se il paziente ha ricevuto un trapianto di midollo, a che distanza di questo si è eseguito il bal [ ] . bronchoalveolar lavage in children sedation with meperidine and midazolam in paediatric patients undergoing endoscopy serum lidocaine concentrations in children during bronchoscopy with topical anestesia comparison of the incidence of complication at induction and emergence in infants receiving oral atropine versus no premedication technical recommendations and guidelines for bronchoalveolar lavage (bal) adjustment of bronchoalveolar lavage volume to body weight in children a simple method of reducing complications of paediatrics nonbronchoscopic bronchoalveolar lavage bronchoalveolar lavage cellularity in infants with severe respiratory syncytial virus bronchiolitis bronchoalveolar lavage in intensive care units complications of fiberoptic bronchoscopy in thrombocytopenic patients proteomics as the tool to search for lung disease markers in bronchoalveolar lavage differential cytology of bronchoalveolar lavage fluid in normal children bronchoalveolar lavage cellularity in healthy children bronchoalveolar lavage studies in children without parenchymal lung disease:cellular constituents and protein levels chronic interstizial lung disease in children lymphocytes subsets in bronchoalveolar lavage fluid of children without bronchopulmonary disease cd a-positive cells in bronchoalveolar lavage samples from children with langherans cell histiocytosis fiberoptic bronchoscopy and bronchoalveolar lavage in the management of children with gastroesophageal reflux bronchoalveolar lavage and esophageal ph monitoring data in children with "difficult to treat"respiratory symptoms recurrent aspiration in cildren:lipid-laden alveolar macrophages quantitation bronchoalveolar lavage abnormalities in children with gastroesophageal reflux and "difficult to treat"respiratory symptoms:correlation with phmetry data il- levels and airway neutrophilia in children with gastroesophageal reflux and asthma-like symptoms diagnosing pulmonary alveolar proteinosis the role of bronchoalveolar lavage in the immunocompromised host use of polymerase chain reaction for improved diagnosis of tuberculosis in children rapid detection of multidrug-resistant tuberculosis semiquantitative detection by real-time pcr of aspergillus fumigatus in bronchoalveolar lavage fluids and tissue biopsy specimens from patients with invasive aspergillosis pulmonary complication in bone marrow transplantation:a practical approach to diagnosis and treatment whole lung lavage pulmonary alveolar proteinosis pulmonary alveolar proteinosis in children valutando le possibilità diagnostiche del bal come sopraesposte, e differenziando i pazienti pediatrici immunocompetenti da quelli immunocompromessi, le indicazioni ad eseguire il bal in questi due gruppi di pazienti possono essere così riassunte.pazienti immunocompetenti: . il bal andrebbe eseguito in ogni paziente che presenti malattia interstiziale polmonare, per poter valutare il tipo di infiammazione (alveolite) in atto ed aiutare così a formulare una diagnosi e porre una terapia; . nel caso poi in cui si sospetti la presenza di una polmonite lipoidea da inalazione di materiale alimentare, come nella malattia da reflusso con sintomi sovraesofagei, o un'emorragia alveolare in atto, in questi casi il bal non solo orienta la diagnosi, ma riveste un ruolo essenziale nel processo diagnostico.pazienti immunocompromessi: eseguire il bal in caso di: . inizio acuto di tachidispnea, ipossiemia e comparsa di infiltrati polmonari: eseguire subito il bal, ancor prima di iniziare la terapia antibiotica; qualora questa sia stata già iniziata, il bal andrà eseguito in quei pazienti che non mostrino miglioramento clinico; . comparsa di un unico infiltrato polmonare, che non risponda dopo ore di terapia antibiotica ad ampio spettro; . nelle polmoniti croniche interstiziali, sia nei pazienti hiv che non hiv. il bal può anche essere impiegato, anche se molto raramente, a scopo terapeutico, per rimuovere da un lobo o da un intero polmone sostanze nocive. queste possono essere sia di origine esogena, come nella polmonite lipoidea da inalazione cronica di oligominerale, sia endogena, come nella proteinosi alveolare (congenita o acquisita).in questi casi, poiché il lobo o il polmone che viene "lavato" viene effettivamente inondato di soluzione fisiologica, viene posizionato un catetere a palloncino prossimamente alla zona di esecuzione del bal, così che gli altri distretti polmonari sono risparmiati. il lavaggio viene eseguito instillando ed aspirando in maniera sequenziale grosse aliquote di soluzione fisiologica ( - ml), continuando la metodica fino a che il liquido aspirato non appaia limpido, ovvero non contenga più lipidi [ ] [ ] [ ] . key: cord- -elzhww a authors: van driessche, l.; valgaeren, b.r.; gille, l.; boyen, f.; ducatelle, r.; haesebrouck, f.; deprez, p.; pardon, b. title: a deep nasopharyngeal swab versus nonendoscopic bronchoalveolar lavage for isolation of bacterial pathogens from preweaned calves with respiratory disease date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: elzhww a background: nonendoscopic bronchoalveolar lavage (bal) is a practical alternative for a deep nasopharyngeal swab (dns) to sample the airways of a large number of calves in a short period of time. the extent of commensal overgrowth and agreement of bal with dns culture results in preweaned calves are unknown. objectives: to compare commensal overgrowth and bacterial culture results between dns and bal samples. animals: a total of preweaned calves ( with bovine respiratory disease and healthy animals). methods: cross‐sectional study. deep nasopharyngeal swab and bal samples were taken from each calf and cultured to detect pasteurellaceae and mycoplasma bovis. agreement and associations between culture results of dns and bal samples were determined by kappa statistics and logistic regression. results: bronchoalveolar lavage samples were less often polymicrobial, more frequently negative and yielded more pure cultures compared to dns, leading to a clinically interpretable culture result in . % of the cases compared to only in . % of the dns samples. isolation rates were lower in healthy animals, but not different between dns and bal samples. only histophilus somni was more likely to be isolated from bal samples. in clinical cases, a polymicrobial dns culture result did not increase the probability of a polymicrobial bal result by ≥ %, nor did it influence the probability of a negative culture. a significant herd effect was noted for all observed relationships. conclusions and clinical relevance: nonendoscopic bal samples are far less overgrown by bacteria compared to dns samples under the conditions of this study, facilitating clinical interpretation and resulting in a higher return on investment in bacteriologic culturing. b ovine respiratory disease (brd) has major economic impact in cattle production systems worldwide. it is the main indication for antimicrobial use in calves and therefore receives considerable attention in countries in which veterinary use of antimicrobials is in question. to rationalize antimicrobial use, veterinary formularies have been established in several european countries such as belgium, the netherlands, and denmark. these formularies recommend sampling of the respiratory tract, bacterial isolation, and susceptibility testing before certain antimicrobial classes, critical for human medicine, can be used. recently, a change in belgian law has been made, requiring an antibiogram before fluoroquinolones or cephalosporins can be used. however, to date, there is no consensus on how the respiratory tract should be sampled to isolate causative pathogens. in practice, deep nasopharyngeal swabs (dns), - transtracheal aspiration (tta), and bronchoalveolar lavage (bal) , have been used for sampling the respiratory tract. deep nasopharyngeal swab is the easiest, fastest, and cheapest technique and therefore most suitable for sampling large numbers of animals. one major disadvantage is that dns does not sample the site of interest (pneumonic lung). previous work in a single feedlot showed moderate agreement between dns and bal culture results in calves for pasteurellaceae (pasteurella multocida, mannheimia haemolytica sensu lato, and histophilus somni) and mycoplasmata. transtracheal aspiration samples the bronchial bifurcation, but has the disadvantage of being more time-consuming, expensive, and invasive, while at the same time holding a certain risk (e.g., hemorrhage, emphysema, infection) for the animal. agreement between dns and tta culture results was reported in fattening bulls to be moderate for m. haemolytica s.l. a bal often is performed with an endoscope, which requires costly equipment and carries high risk of contamination when sampling multiple animals successively. alternatively, bal can be performed with a reusable sterilized bal catheter without endoscopic guidance. this makes it easier for large numbers of animals to be sampled at the lung level in a short time frame and with a low cost per calf. however, an important point of criticism is the nasal passage of the bal catheter, which may inoculate the bal sample with either respiratory pathogens of the nasal cavity or commensal microflora. despite the high prevalence of brd in preweaned calves, , information on the performance of nonendoscopic bal and the agreement of dns and bal culture results in preweaned calves currently is not available. results in preweaned calves might substantially differ from those in feedlot cattle, because preweaned calves are more likely to suffer from their first brd episode, whereas the older feedlot cattle might relapse, and residual pathogenic flora in the lung might differ from the dominant nasopharyngeal flora. therefore, the objectives of our study were ( ) to determine the outcome of bacterial culture results, isolation rates, and agreement for samples taken with dns and nonendoscopic bal with respect to pasteurellaceae and mycoplasma bovis. infections in preweaned calves; ( ) to determine the polymicrobial nature of dns and bal samples; and ( ) to determine whether a polymicrobial dns culture result, caused by the nasopharyngeal flora or unhygienic sampling, influences bal culture results. all sampling techniques and the study protocol were revised by the local ethical committee and permitted under experimental license number ec - . sample size was calculated to detect a % difference in culture results (i.e., prevalence of pure cultures) between dns and bal samples in calves with brd (cases) and controls with % confidence and % power. required sample size for a -sided test was observations per group. a the sample size for the cases was increased . times to increase the probability that all major brd pathogens would be present in the data set. a cross-sectional study was performed on commercial herds ( veal, beef) between september and may . the study was divided into parts. in herds, animals with clinical brd (cases) were sampled, and in ( veal and beef) herds, only healthy animals were sampled (controls). veal calves were group-housed ( - ) on a slatted floor and fed milk replacer, concentrates, and roughage according to european legislation (ec - ). beef calves also were group-housed ( - calves per group) on straw and received milk replacer, concentrates, and roughage. the herds with clinical brd were reported by local veterinarians and subsequently visited by the research staff. calves to be sampled (cases) were selected based on previously described inclusion criteria. briefly, the following clinical signs were scored on a -point scale (score - ): lethargy (from standing to recumbency and position of the ears), cough (from absent to spontaneous), rectal temperature (from < °c to > . °c), and nasal discharge (from absent to bilateral purulent). an animal with a score ≥ was considered a case, independent on how many clinical signs were abnormal. additionally, thoracic ultrasound examination was performed with a . -mhz linear probe b as previously described. the definition for a case was the presence of a consolidated zone in the lung of ≥ cm . in the affected herds, all animals that met the inclusion criterion were sampled. to avoid subclinical infection or inflammation (bronchitis-pneumonia) because of exposure to brd risk factors, controls were selected from herds that had not experienced a brd outbreak in the last month. controls had to have a normal clinical investigation ( on the -point scale) and absence of any ultrasonographic abnormalities. animals that were vaccinated against brd or treated with antimicrobials days before sampling were excluded from the study. from each calf, an unguarded dns and then a bal sample were taken as previously described. before inserting a dns, the animal was restrained while standing and the nostrils were disinfected with % alcohol. a -cm sterile transport swab c was used. the swab was sufficiently long to cover the distance from the nostril to the medial canthus of the eye, hereby sampling nasopharyngeal tissue. the swab was introduced medioventrally in the nasal cavity until the nasopharyngeal tissue was reached. after rotating several times, the swab was taken out and placed in amies transport medium without charcoal formulas. bronchoalveolar lavage fluid was collected by a reusable homemade polytetrafluorethylene catheter d adjusted with a -g catheter stylet. the procedure was performed in standing animals without sedation as previously described. briefly, after rinsing the nostril with % alcohol, the catheter was inserted medioventrally in the nasal cavity, passed through larynx and trachea, and gently advanced into the bronchi until the wedge position was reached. next, ml of sterile . % nacl was injected into the lungs and immediately aspirated (recovery of - % of the fluid). if no fluid was recovered, a second ml injection was attempted. sample validity was checked by inspecting for the presence of the characteristic foam layer, indicating contact with surfactant. samples were transported at ambient temperature and cultured within hours after sampling. for each calf, a new sterilized catheter was used. sampling was performed by different veterinarians ( - different samplers per herd, different samplers in total). deep nasopharyngeal swab and bal samples ( . ml) were inoculated on columbia blood agar e enriched with % sheep blood and on pleuropneumonia-like organism (pplo) agar ( . g d-glucose and g pplo f in ml of distilled water [ph = . - . ]) for isolation of pasteurellaceae and m. bovis, respectively. blood agars were incubated overnight and pplo agars for days, both at °c and % co . bacteria were selected based on phenotypic characteristics and subsequently further identified by biochemical tests according to as previously described. identification of m. bovis was made by culturing on pplo agar enriched with polysorbate . mycoplasma bovis colonies showed the typical "fried-egg" morphology on microscopic examination. if no growth was observed after this period, incubation was continued for h for pasteurellaceae and days for m. bovis. all bacteriological analyses were performed at the department of bacteriology at the faculty of veterinary medicine, gent university, belgium. culture results were interpreted as follows: a negative culture result was defined as the absence of growth of the target bacteria or the presence of < colonies of contaminants after h of incubation for pasteurellaceae. a polymicrobial result was defined as the growth of multiple bacterial colonies with different morphologies on the agar of which no target bacteria could be subjected to subculture for further identification. a pure culture result was defined as the presence of bacterial species on the agar (> colonies). the presence of several (< ) bacterial species on the agar with dominant growth of species was defined as a dominant culture. isolation rates of the studied bacteria were calculated by dividing the sum of pure and dominant cultures (i.e., positive cultures) by the total number of samples. all results, except for polymicrobial results, were considered clinically interpretable. the experimental unit was the individual calf. to compare isolation rates between dns and bal samples, a multivariable linear mixed model was constructed (proc glimmix) with the respective bacteriological result (e.g., p. multocida or pure culture) as the outcome variable and swab/bal as a binary variable factor. a binomial distribution and logit link function with wald's statistics for type contrasts was used. herd was added as a random factor to account for clustering. no agreement was investigated among the different veterinarians involved. agreement between dns and bal for the isolation of p. multocida, m. haemolytica s.l., h. somni, and m. bovis was determined by means of the kappa statistic. strength of agreement for the kappa coefficient was interpreted as previously described (≤ = poor; . - . = slight; . - . = fair; . - . = moderate; . - . = substantial; and . - . = almost perfect). the association between isolation of a bacterial species from the dns sample and its isolation from the bal sample was determined by means of a multivariable linear mixed model (proc glimmix). eight different models were constructed, separate for cases and controls, with the respective pure culture (m. haemolytica s.l., p. multocida, m. bovis, and all pure cultures), a polymicrobial culture, dominant culture, or negative result as the outcome variables. a binomial distribution and logit link function with wald's statistics for type contrasts was used. herd was added as a random factor to account for clustering. to determine the effect of a polymicrobial dns culture result on the probability of a pure culture in the bal sample in calves with brd, different general linear mixed models were constructed with m. haemolytica s.l., p. multocida, m. bovis, and a negative culture result as outcome variables. the same procedure as described above was followed. model validity was evaluated by the hosmer-lemeshow goodness-of-fit test for logistic models. significance was set at p < . . all analyses were performed in sas . . g details on herd types, number of animals sampled, and sampling results at herd level are provided in table . mannheimia haemolytica s.l., p. multocida, and h. somni were found in . % ( of ), . % ( of ), and . % ( of ) of the brd outbreak herds, respectively. mycoplasma bovis was only found in both veal farms with brd outbreaks ( . %; of ). very few targeted respiratory pathogens (n = ) could be retrieved from the control herds. in herds (herds and ), p. multocida isolates were retrieved, whereas in herd , h. somni isolates also were retrieved. in herd , m. bovis was isolated from a single calf. in herd , no respiratory pathogens could be isolated ( table ). with dns and bal, both in cases as controls, p. multocida (n = ) was isolated most frequently, followed by m. bovis (n = ), m. haemolytica s.l. (n = ), and h. somni (n = ). in case calves, the isolation rates were not significantly different between dns and bal for all studied bacteria, except for h. somni which was less frequently isolated from dns (p < . ; table ). mixed infections (i.e., isolation of ≥ respiratory target bacteria from the same dns or bal sample) were only seen in cases from the veal farms (table ). in cases, agreement between dns and bal culture results was moderate for all bacteria (j = . - . ), with the exception of h. somni, for which it was slight (j = . ; table ). a positive dns culture result in cases significantly increased the odds of a positive bal for m. haemolytica s.l., p. multocida, and m. bovis (table ). this relationship was significantly affected by the herd effect (p < . ). case beef - ( ) ( ) ( ) ( ) ] in controls), meaning that no pasteurellaceae or m. bovis could be phenotypically identified from the plate. compared to dns, bal samples were significantly less polymicrobial (p < . for cases and controls), more often negative (p < . for cases, p < . for controls), and more often returned pure cultures of pasteurellaceae or m. bovis (p < . for cases, p < . for controls; table ). in summary, bal samples returned an interpretable result (either negative, pure, or dominant culture result) in . % of the cases and in . % of the controls, compared to . % and . % for dns in cases and controls, respectively (p < . for both comparisons; table ). the polymicrobial nature of a sample result was strongly affected by the herd effect (p < . ). a polymicrobial dns and bal culture result in at least animal was present in almost all herds ( of herds, the other herds had no polymicrobial bal culture result), but there was very large variation in the percentage of polymicrobial results among the herds sampled (table ). in the cases, a polymicrobial dns culture result did not increase the probability of a polymicrobial bal result by ≥ % (p = . ), nor did it influence the probability of a negative culture (p = . ). however, the probability of retrieving m. haemolytica s.l. and p. multocida from the bal sample still decreased when the dns was polymicrobial. in contrast, there was no effect of a polymicrobial dns result on the probability of isolation of m. bovis from the bal sample (table ). to determine how the respiratory tract should be sampled to isolate the causative pathogens, a crosssectional study was performed to compare bacterial culture results and commensal overgrowth between dns and bal samples. sampling procedures returning high isolation rates of the major respiratory pathogens and with a straightforward interpretation of the culture results have the highest return on investment and are therefore most suitable for practice. in our study, all isolates were identified by biochemical tests and morphology instead of by polymerase chain reaction (pcr). this approach might limit the results with respect to bacterial species identification. biochemical identification was selected because it is the routine identification method used in private laboratories in belgium and neighboring countries, for reasons of speed and cost of analysis. the objective of our study was to gain insights into the sampling and culture methods currently used in the field. also, no selective media to increase pasteurellaceae isolation rates were used, because doing so currently is not the standard procedure used in private laboratories. selective media would likely decrease contamination, whereas of the main objectives was to study differences in contamination between dns and bal. a final limitation of this study was that, for practical reasons, the returned lavage fluid volume was not determined. quantification of the target bacteria was not an objective of the study, but differences in the returned volume might potentially have influenced culture results. one of the main findings in the study on preweaned calves is that isolation rates of respiratory bacterial pathogens in both dns and bal samples were lower in controls compared to cases. the most likely herd effect was significant for all studied outcomes, except p. multocida. explanation is that the control group consisted of animals originating from other farms than the case farms, whereas in previous work, "apparently healthy" incontact animals were used as controls. these apparently healthy animals are likely exposed to the same risk factors as the cases and might be subclinically infected. therefore, in our study, controls were deliberately chosen from farms without recent brd exposure, and ultrasound examination was used as an additional tool to aid in selecting truly healthy animals. a disadvantage of this approach is the environmental differences (e.g., bedding, herd size, air quality) that exist among herds. to definitively determine whether isolation rates differ between diseased and truly healthy animals in herd, a longitudinal study design would be needed. agreement between dns and bal samples was moderate for m. haemolytica s.l., p. multocida, and m. bovis, similar to what was observed for m. haemolytica s.l. in fattening bulls. agreement was much lower for h. somni, which can be explained by the fact that h. somni is easily overgrown by other bacteria. given their polymicrobial nature, dns samples are likely to be falsely negative for h. somni, when no selective media are used. current understanding of the pathogenesis of bacterial pneumonia in calves suggests overgrowth of pasteurellaceae in the nasopharynx and tonsils with subsequent colonization of the trachea and lungs. even when applying a transtracheal sampling procedure, in diseased animals, one is probably as likely to isolate bacteria that have descended from the nasopharynx as those originating from the lung. possible reasons why dns and bal samples do not agree are false-negative results caused by polymicrobial overgrowth (sampling technique or presence of resident flora), sampling of a nonaffected lung lobe with the nonendoscopic bal technique, or the absence of deep bronchitis or alveolitis in case calves. the latter reason was excluded as much as possible by the use of ultrasound examination in this study. previous work showed that this nonendoscopic bal approach samples a random lung lobe in nonsedated animals, and not necessarily the most frequently affected cranial lobes. this might in part explain why some cultures of cases were negative. however, we doubt this is true, and our hypothesis is that passage through trachea and deep bronchi transfers bacteria deeper into the lung. interestingly, in the same animal, the dns could be polymicrobial, whereas the bal yielded a pure culture, dominant culture, or even a completely negative result. additionally, the polymicrobial nature of the dns did not affect the presence of a negative or pure culture result in the bal. also, h. somni could be isolated in pure culture from the lungs of diseased calves, whereas it was overgrown or absent on the nasopharyngeal culture. these observations strongly suggest that, under the conditions of our study, nasopharyngeal contamination of a bal sample is less common than previously assumed. to what extent a possible cleansing effect of the dns contributes to a pure culture result in the bal is unclear. on the other hand, a dns polymicrobial result did decrease the probability of isolating m. haemolytica s.l. or p. multocida from the bal, whereas this effect was not observed for m. bovis for which selective media were used. again, this observation could be explained by bal placement in a healthy lung lobe in a case calf or because respiratory bacteria are not necessarily involved in every case. several viruses (e.g., bovine respiratory syncytial virus, bovine coronavirus) are capable of inducing pneumonia and marked disease without bacterial superinfection. unfortunately, in our study, viral analysis in each case was not possible for financial reasons. however, in our opinion, the polymicrobial nature of dna and bal is strongly influenced by the sampling (technique and dns, deep nasopharyngeal swab; bal, bronchoalveolar lavage; nd, no statistical analysis possible, because of a too small number of observations in one of the groups; or, odds ratio; ci, confidence interval. the random herd effect was significant in all models. hygiene), given that such a strong herd effect on the sampling results was observed. to overcome the issue of possible nasopharyngeal overgrowth in dns and bal samples due to nasopharyngeal passage, both the use of selective media for isolation of pasteurellaceae (e.g., addition of bacitracin , ) and a more quantitative approach to bal results might be suitable. our study focused on culture results obtained when applying dns and bal as in practice. to definitively determine the extent and diagnostic importance of possible overgrowth as a consequence of nasopharyngeal passage, experimental work with intensive strain typing and necropsy to confirm the infective status of the lung will be needed. as mentioned above, a significant herd effect was noted on many of the outcomes studied. deep nasopharyngeal swab and bal were performed only after cleaning the outer nares and without a protective sleeve as used in previous studies. , this could have increased the risk of contamination by bacteria residing in the nostril. in belgium, dns for practical reasons is routinely performed without a protective sleeve, again increasing external validity in this study. multiple samplers participated in the study, and although all of them received at least training session from the same trainer before the start of the study, variation in the extent of experience in taking dns or bal samples and in the hygienic procedures accompanying these techniques might have influenced the results. deep nasopharyngeal swab samples might be polymicrobial due to the presence of a highly variable nasopharyngeal microflora or due to environmental contamination (e.g., touching the muzzle or other objects during sampling). other reasons might be environmental or aerosolized dust, endotoxin, bedding conditions, and issues with stable ventilation. likely, the risk of catheter contamination increases when repeated attempts to enter the trachea are needed or when the esophagus is accidently entered. adequate training is likely the only solution, other than considering other procedures such as protective sleeves, agar plugs, or visualization of the larynx through a low-cost laryngoscope. in conclusion, a nonendoscopic bal results in less contaminated (and therefore more easily interpretable samples) compared to dns under the conditions of this study. it returns an interpretable result in . % of the cases, compared to . % in dns, and has better isolation rates for h. somni, offering a better return on investment for bacteriological sampling. it can be performed rapidly in a representative number of animals at low cost and likely has less impact on animal welfare then more invasive techniques. bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors prospective study on quantitative and qualitative antimicrobial and anti-inflammatory drug use in white veal calves monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands koninklijk besluit betreffende de voorwaarden voor het gebruik van geneesmiddelen door de dierenartsen en door de verantwoordelijken van de dieren variability in acquired resistance of pasteurella and mannheimia isolates from the nasopharynx of calves, with particular reference to different herd types use of deep nasopharyngeal swabs as a predictive diagnostic method for natural respiratory infections in calves prevalence of respiratory pathogens in diseased, non-vaccinated, routinely medicated veal calves transmission dynamics of mannheimia haemolytica in newly-received beef bulls at fattening operations bronchoalveolar lavage of cranial and caudal lung regions in selected normal calves: cellular, microbiological, immunoglobulin, serological and histological variables comparison of sampling procedures for isolating pulmonary mycoplasmas in cattle the microbial flora of the respiratory tract in feedlot calves: associations between nasopharyngeal and bronchoalveolar lavage cultures vergleichende auswertung der bakteriologischen untersuchungsbefunde von nasen-und trachealtupfern sowie trachealsp€ ulproben assie s, seegers h, beaudeau f. incidence of respiratory disorders during housing in non-weaned charolais calves in cowcalf farms of pays de la loire prediction of respiratory disease and diarrhea in veal calves based on immunoglobulin levels and the serostatus for respiratory pathogens measured at arrival short communication: ultrasonographic assessment of the thorax as a fast technique to assess pulmonary lesions in dairy calves with bovine respiratory disease the mycoplasmas (class: mollicutes) a coefficient of agreement for nominal scales an application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers haemophilus species pasteurella haemolytica in the tracheal air of calves estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution changes in the bacterial flora of the upper and lower respiratory tracts and bronchoalveolar lavage differential cell counts in feedlot calves treated for respiratory diseases we thank karlijn janssens and pieter de wolf for sampling some of the calves. all practitioners are acknowledged for reporting suitable outbreaks for this study.conflict of interest declaration: authors declare no conflict of interest.off-label antimicrobial declaration: authors declare no off-label use of antimicrobials. key: cord- -t dtabi authors: bousbia, sabri; papazian, laurent; saux, pierre; forel, jean marie; auffray, jean-pierre; martin, claude; raoult, didier; la scola, bernard title: repertoire of intensive care unit pneumonia microbiota date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: t dtabi despite the considerable number of studies reported to date, the causative agents of pneumonia are not completely identified. we comprehensively applied modern and traditional laboratory diagnostic techniques to identify microbiota in patients who were admitted to or developed pneumonia in intensive care units (icus). during a three-year period, we tested the bronchoalveolar lavage (bal) of patients with ventilator-associated pneumonia, community-acquired pneumonia, non-ventilator icu pneumonia and aspiration pneumonia, and compared the results with those from patients without pneumonia (controls). samples were tested by amplification of s rdna, s rdna genes followed by cloning and sequencing and by pcr to target specific pathogens. we also included culture, amoeba co-culture, detection of antibodies to selected agents and urinary antigen tests. based on molecular testing, we identified a wide repertoire of bacterial species of which have not been previously reported in pneumonia. moreover, we found putative new bacterial phylotypes with a s rdna gene divergence ≥ % from known phylotypes. we also identified fungal species of which have not been previously reported in pneumonia and viruses. patients can present up to different microorganisms in a single bal (mean ± sd; . ± . ). some pathogens considered to be typical for icu pneumonia such as pseudomonas aeruginosa and streptococcus species can be detected as commonly in controls as in pneumonia patients which strikingly highlights the existence of a core pulmonary microbiota. differences in the microbiota of different forms of pneumonia were documented. the cause of pneumonia in intensive care units (icus) remains unknown in nearly % of cases despite extensive microbiological investigations [ , ] . microbial communities previously identified, in deep respiratory samples, include bacteria, fungi and viruses for which the role in the observed pathology is not clear. microorganisms frequently identified in respiratory samples from icupneumonia patients included pseudomonas aeruginosa, staphylococci, enterobacteria, candida albicans, influenza virus, herpes simplex virus (hsv) and cytomegalovirus (cmv) [ ] [ ] [ ] [ ] [ ] . in some investigations, a pathogenic bacterium is isolated, whereas in other cases, the number of colony forming units (cfu) is considered to determine the pathogenic character [ ] . recently, the bacterial microbiota of patients with cystic fibrosis and ventilator-associated pneumonia (vap) were studied using s rdna gene amplification followed by clone libraries sequencing [ ] [ ] [ ] . our laboratory has contributed to this work and has shown, by different sequencing approaches, that the microbial population of patients with cystic fibrosis was more diverse than expected [ , ] . here, we use a comparable approach in order to study episodes of icu pneumonia and control cases. these patients were studied using broad-range primer amplification of the s rdna gene of bacteria and the intergenic spacer of s rdna gene of fungi followed by cloning and sequencing. we also used specific quantitative pcr (qpcr) to target fastidious bacteria and a spectrum of viruses. moreover, we tested samples from our patients by standardized routine culture, amoebal co-culture, blood culture, elisa targeted antibody detection, immunofluorescent assay antigenemia and antigenuria testing as routinely performed in such cases to compare these routine tests with molecular approaches. in preliminary results, we have reported the likely frequency of tropheryma whipplei and the occurrence of vegetable dna in pneumonia patients [ , ] . in this work, we highlight the different compositions of microbiota in patients with four different types of icu-pneumonia. bacterial microbiota as evaluated by s rdna molecular assays were positive for at least one bacterium for out of bronchoalveolar lavage (bal) samples from patients with pneumonia as well as from out of from control individuals (p = . ). bacterial clone libraries from amplified s rdna genes (nearly , clones that contained exploitable sequences were included) identified different bacteria at the species level. detailed data about the relative abundance and richness of each species in their corresponding library are summarized in data s and s in supplementary informations. bacterial clone libraries of patients showed that libraries were characterized by the presence of only one bacterium, libraries were polybacterial but dominated by one bacterium ( % of the clones in the library), whereas libraries were polybacterial without any dominant bacterium (fig. ) . bacterial clone libraries of controls showed that libraries were characterized by the presence of only one bacterium, libraries were polybacterial but dominated by one bacterium ( % of the clones in the library), whereas libraries were polybacterial but without any dominant bacterium (fig. ) . patients exhibited up to bacteria in their bal fluids (mean sd; . . ) (tables and ). overall, patients exhibited different species belonging to different phyla ( classes, orders, families and genera) of which had not been previously observed in bal from pneumonia, whereas bacterial clone libraries of controls identified species belonging to different phyla ( classes, orders, families and genera). in patients, aerobic gram-negative bacilli, gram-positive cocci, and anaerobic bacteria from oropharyngeal flora were the most frequent bacteria identified (tables s ,s ,s ,s , fig. ) . surprisingly, bacteria that are usually associated with other diseases such as the gram-positive anaerobe atopobium vaginae, or from unexpected animal origins, such as enterococcus canintestini, were alsoc found. moreover, strictly anaerobic bacteria ( %) were found in patients versus anaerobic bacteria ( %) found in controls (p = . ). among those bacteria which were identified in controls, bacteria were also identified in patients (fig. s , tables s ,s ,s ), including pseudomonas aeruginosa sequences respectively identified in % and % from different clones libraries from immunocompromised controls. in the second clone library, the % of the remaining sequences included achromobacter xylosoxidans, which also is a typical bacterium of nosocomial pneumonia. stenotrophomonas maltophilia sequences were found in % of clone library of another immunocompromised control, along with other bacteria. similarly, sequences of streptococcus mitis ( % of the clone library) was identified along with other bacteria in a control with acute respiratory distress syndrome (ards) and a history of aspiration pneumonia (ap) days before bal sampling. additionally, arcobacter cryaerophilus, atopobium parvulum, lachnospiraceae bacterium, prevotella melaninogenica, and prevotella pallens were significantly more frequent in controls than in patients (p = . , . , . , . and . respectively) (table s ) . bacterial clone libraries surprisingly showed new phylotypes with s rdna sequence similarity lower than % to known bacteria available in the genbank database (data s and s ). among them, novel bacterial phylotypes were identified in bal from patients, whereas novel phylotypes were identified in bal from controls (fig. ) . novel bacterial phylotypes identified in patients were more diversified, as they belonged to different phyla including bacteroidetes ( phylotypes), firmicutes ( phylotypes), proteobacteria ( phylotypes), actinobacteria (one phylotypes), acidobacteria (one phylotype) and spirochaetes (one phylotype). novel species identified in controls belong to bacteroidetes ( phylotypes), firmicutes ( phylotypes) and actinobacteria (one phylotype). prevotellaceae phylotypes represent % of all novel phylotypes identified and they were exhibited in patients with pneumonia as well as in control subjects (fig. ) . results obtained using routine bal and blood culture are available text s . fungal microbiota as evaluated by the intergenic spacer of s rdna at least one fungus was found in bal patient samples and in from controls (p = . ). positive patients exhibit up to fungi in their bal fluids (mean sd; . . ) (tables and ) . detailed data about the relative abundance and richness of each species in their corresponding library are also summarized in summary files (data s and s ) in supplementary informations. fungal microbiota obtained from patients showed the presence of different species belonging to phyla ( orders, families and genera) among which phylotypes had not been previously identified in bal fluids from pneumonia. clone libraries from controls, identified fungi belonging to one phylum ( orders, families and genera) among which fungi were also identified in patients. candida species were the most common fungal species identified (tables s ,s ,s and s ). environmental fungi, which usually colonize water, food debris and humid building surfaces, were more notably identified in our study than in previous pneumonia studies. furthermore, tree fungi belonging to basidiomycota phylum, sporidiobolales sp., cryptococcus victoriae and hyphoderma praetermissum, were found for the first time in pneumonia bal samples in the present study, while candida utilis and periconia macrospinosa were identified only in controls. additionally, candida utilis was significantly more frequent in controls than in patients (p = . ). results of fungi obtained using a routine bal culture are available in text s . four pneumonia patients were found to be positive for fastidious bacteria chlamydia pscitacii ( case), mycobacterium sp. ( cases) and mycoplasma pneumoniae ( cases) by qpcr. in addition, qpcr enabled the detection of different viruses. quantitative data of microorganisms identified by qpcr (loads or cycle threshold) are also provided and summarized in supplementary information (data s and s ). our study showed that at least one virus was identified in bal samples from patients, and from controls (p = . ). hsv and cmv were the most commonly identified viruses. while the prevalence of these two viruses in patients was not significantly different from that of controls (table s ) , cmv was more frequently identified in pneumonia patients than in controls. hsv and cmv coinfection was found in bal samples from vap patients, community-acquired pneumonia (cap) patients and non-ventilator icu pneumonia (nv icu-p) patients and one control subject. coinfection with cmv and respiratory syncytial virus type a was detected in a bal from one nv-icu-p patient, and both hsv and vzv were identified in a bal from a cap patient. rhinovirus was identified in a control with ards, urinary infection and sinusitis. parainfluenza virus- was detected in vap patients and an immunocompetent control with a pulmonary contusion. results obtained using routine serology and antigenemia for viruses and fastidious pathogens are available in text s overall, bacterial difference between patients and controls showed that bacteria belonging to bacilli and gammaproteobacteria were dominant in patients, whereas anaerobic bacteria related to bacteroidia (represented essentially by prevotella species) and clostridia were dominant in controls ( fig. ) (p, . ). mollicutes, which are represented by the mycoplasma genus, were only detected in patients with cap and vap (fig. ) . as for fungal species, members of saccharomycetes were ubiquitously identified in all cohorts. eurotiomycetes, which are represented by aspergillus, penicillium and cladophialophora genera, were dominant in the cap cohort (fig. ) . tremellomycetes, represented by the cryptococcus genus, was only identified in the nv-icu-p cohort, whereas figure . a phylogenetic tree inferred from s rdna sequences of novel bacterial phylotypes. these novel phylotypes exhibited sequence similarities of less than % to known bacteria available in the genbank database, and they were classified in silico using ''classifier'' program. phylotypes are reported according to their genus or by the last possible classification determined by the program. when possible, phylotypes with the same classification are clustered together. the frequency of phylotypes in each cohort is shown on the right.. bacteroidetes are shown in purple, firmicutes in red, proteobacteria in blue, actinobacteria in yellow, acidobacteria in orange and spirochaetes in green. cap, community-acquired pneumonia; vap, ventilator-associated pneumonia; nv icu-p, non-ventilator icu pneumonia; ap, aspiration pneumonia; and cs, control subjects. doi: . /journal.pone. .g agaricomycetes and an unclassified ascomycota (melanized limestone ascomycetes) were only identified in the vap cohort. in addition, sordariomycetes, which is represented by the periconia genus, was only identified in controls. at the specie-level, bacteria, fungi and viruses were common to at least two cohorts, among which pseudomonas aeruginosa, streptococcus mitis, prevotella melaninogenica, peptostreptococcus stomatis, candida albicans and hsv were commonly identified irrespective of cohorts, whereas haemophilus influenzae, staphylococcus aureus, streptococcus genomosp. c , streptococcus parasanguinis and streptococcus pneumoniae were commonly identified in patients regardless of pneumonia type (fig. s ) . additionally, bacteria, fungi and viruses were common to controls and at least one pneumonia cohort, whereas bacteria, fungi and viruses were common to at least two pneumonia cohorts (fig. s ). in contrast, many microorganisms ( bacteria, fungi and viruses) were restricted to one of cohorts ( bacteria and fungi only were identified in the cap cohort; bacteria and fungi only were identified in the vap cohort; bacteria, fungi and one virus only were identified in the nv icu-p cohort; bacteria only were identified in the ap cohort; bacteria, fungi and one virus only were identified in controls) (fig. s ). microbial profiles of positive pneumonia bal fluids showed that ( %) were characterized by the presence of one microorganism, whereas ( %) were polymicrobial. in controls, ( %) of bal fluids were characterized by the presence of one microorganism, whereas ( %) were polymicrobial (data s and s ). available clinical data for patients and controls showed that monobacterial patients were more frequently, but statistically insignificant, subjected to initial antibiotic therapy than polymicrobial ones (p = . ; table and table s ). in ventilated subjects, monomicrobial patients have a slightly shorter period of mechanical ventilation prior to the pneumonia episode as compared to polymicrobials. monomicrobial controls have a remarkably shorter period of mechanical ventilation before sampling compared to polymicrobials (p = . ; table ). the same observation was showed for length of icu stay prior to sampling and for total length of hospital stay. according to these observations, the polymicrobial profiles of controls seem to be partially related to the high duration of icu stay before sampling. however, the icu mortality was higher in monomicrobial patients than in polymicrobial ones (p = . ). the icu mortality rate was higher in pneumonia patients for whom bal fluids exhibited only viruses or fungi, or both than in monobacterial or polybacterial patients (p = . ; table s ). a higher but not statistically significant icu mortality was also observed in pneumonia patients for whom bal fluids exhibited only viruses or fungi, or both than in controls with the same profile (p = . ; table s ). we next compared the bacterial communities found in our study to those found in lung specimens in five previous studies which were based on s rdna amplification [ , , [ ] [ ] [ ] . comparative analyses of lung microbiota between these studies showed that different genera were found in all of them. among these genera, genera are widely distributed within the studies including gemella, haemophilus, megasphaera, neisseria, porphyromonas, prevotella, pseudomonas, staphylococcus and streptococcus genera which have commonly been found irrespective of study. in contrast, genera were restrictively identified across the studies (table ) . however, at the species level (only the studies that determined bacterial species were included [ , , ] ), comparative analyses showed that from bacteria commonly distributed within the studies, escherichia coli, haemophilus influenzae, prevotella oris, pseudomonas aeruginosa, staphylococcus aureus and streptococcus mitis were commonly found in the four studies. in contrast, bacteria were restrictively identified across the studies (fig. s ) . consequently, comparative analysis at the specie-level showed that some bacteria, such as pseudomonas aeruginosa and staphylococci, are commonly found in pulmonary specimens. however, the pattern of distribution of many other species is distinctly heterogeneous and depending on the specific study and disease. variation of lung microbiota, from one individual to another and from one study to another, suggests that the repertoire of microorganisms associated with respiratory infections still remains incompletely understood. previous studies performed on respiratory specimens showed that unexpected bacteria are increasingly identified, as well as studies describing isolated cases of respiratory infection due to an unexpected bacterium that was detected using molecular techniques [ , , , [ ] [ ] [ ] [ ] . this study extends the analyses to bacteria, fungi and viruses in a large population of icu pneumonia using comprehensive molecular testing. our results demonstrate that nearly % of the microbial species found had not been previously reported in lung samples from pneumonia. therefore, the composition of icu-pneumonia microbiota is more complex, more extensive and more diverse than originally expected. however, we raise the question on the actual role of these microorganisms in pneumonia. indeed, our study reveals that some pathogens that till now had been considered typical for icu pneumonia, such as pseudomonas aeruginosa and streptococcus species, or viruses, such cmv and hsv, can be detected as commonly in controls as in patients (fig. s and s ). this result is emphasized by more recent studies by erb-downward et al. and hilty et al. who showed that a community of lung-resident bacteria including pseudomonas and streptococcus genera can be identified in patients with chronic obstructive disease or asthma, as well as in healthy people [ , ] . our study agrees with the recent literature and highlights the existence of a core pulmonary microbiota, confirming the non-sterility of the lung [ , ] . more interestingly, we showed that pulmonary microbiota heterogeneity can be observed between patients and controls, among pneumonia cohorts and among patients within the same cohort. high pulmonary microbiota heterogeneity was also observed between our study and other previous works performed on cystic fibrosis or vap [ , , ] (fig. s ) . we found that some bacteria were commonly identified in all studies, whereas many others were only identified in one study, and most of these were unexpected. consequently, lung microbiota can vary greatly between individuals, depending on underlying diseases, habits and geographic origin. additionally, these unexpected microorganisms may explain a lack of response to drug therapies in some pneumonia patients. therefore, the possible extension of empiric treatments to cover a large spectrum of microorganisms, especially for patients who do not respond adequately to initial treatment, is questionable. another interesting observation was that mixed infection was observed in many bal fluids from pneumonia patients. interestingly, recent works report that probable interactions between parasitic species can occur in their host, and these reports also show that infection with a given microorganism may increase or decrease susceptibility to infection by another one or can create a cross-immunity response [ ] [ ] [ ] [ ] [ ] [ ] [ ] . such interaction remains to be investigated. moreover, by comparing molecular testing to standard routine methods, this study reveals that many pneumonia-associated pathogens are fastidious or uncultured and highlights a wide discrepancy between culture and molecular microorganism repertoire. our study also shows that the molecular assay remains a more efficient method to detect microorganisms in the pneumonia samples, independently of atmospheric conditions and medium nutrient supplements, which are particularly important for culture, especially for fastidious microorganisms. in addition, microorganism diagnosis was obtained for ( %) episodes of pneumonia by molecular tools compared with ( %) pneumonia episodes for which microorganism diagnosis was successfully done by culture (table s ). in particular, molecular tools seem to be far more sensitive than culture for bacterial detection. this observation is based on the high number of microorganisms, especially bacteria which were identified by molecular methods compared with those detected by culture. in fact, standard and special bal cultures identified few, essentially easily-grown and strictly aerobic or facultative anaerobic bacteria ( species) compared to molecular tools which identified bacterial species (p, . ) (fig s and s ). molecular tools enabled the identification of unexpected bacteria which usually colonize vaginal tracts, such as atopobium vaginae and peptoniphilus lacrimalis, or of other bacteria coming from unexpected animal origins, such as chlamydia pscittasi, enterococcus canintestini and streptococcus bovis, or of potentially known to be associated with other diseases, such as tropheryma whipplei, which were not identified by culture. furthermore molecular tools allowed the detection of pathogenic bacteria such as mycobacterium sp. and mycoplasma pneumoneae, for which identification attempts by culture using specific media were failed due to culture biases. moreover, all bacteria that were first associated with pneumonia in the present study were exclusively identified by molecular methods. these findings are coherent to results from previous studies on bacterial communities of respiratory diseases, including pneumonia, which showed that molecular assays are more sensitive than culture [ , , ] . however, although molecular approaches identified more fungal species than culture, fungal diagnoses were positive for ( %) episodes of pneumonia by culture compared with ( %) pneumonia episodes for which fungal diagnosis was successfully obtained using molecular tools. thus, fungal bal culture was more sensitive to detect some cultured fungi, such as candida species, than molecular approaches. another important finding was the high number of novel bacterial species never previously described to date (bacteria with blastn similarity less than %). this result is concordant with similar studies of pneumonia and cystic fibrosis subjects [ , ] and shows that in respiratory infections, more complex bacteria populations can exist, among which novel bacteria had never been previously identified. moreover, this finding was also supported by other studies performed on endodontic infections, demonstrating that many novel bacteria essentially resident in the oropharyngeal and dental plaque flora can be detected in these infections [ , ] . the oropharyngeal and dental plaque flora is potentially suspected to be a reservoir and, thus, the source of icu pneumonia pathogens, which could suggest that these novel bacteria were inhaled through oropharyngeal tracts [ , ] . nevertheless, molecular tools alone cannot give positive results in some cases, or they could just identify microorganisms known to be commensal or less pathogenic, where it may be useful to perform other tests, such as serology. this was the situation for pneumonia patients for whom serology provided evidence for influenza a virus infection, whereas qpcr performed on their bal fluids was negative. moreover, by combining culture-based methods, blood culture and serology to molecular approaches we significantly increase the probability to detect microorganisms in the pneumonia episodes. in fact, by using these exhaustive laboratory diagnostic tools, we failed to identify a microbial agent in only % of the pneumonia episodes, which is significant when compared to previous studies where the microbial agent was not found in more than % of episodes of pneumonia (p, . ) [ ] . however, the clinical significance of these microorganisms and their role in the etiology of pneumonia remain difficult to be cleared as their correlation with the disease causation remains to be studied and confirmed in the future. nevertheless, our findings suggest that it would be highly recommended to develop a rapid molecular test to target, besides typical pathogens, potential pathogens known to be fastidious or uncultured (such as anaerobic ones), and that it would be useful to add it to existing routine standard techniques. in summary, our study reveals that the respiratory microbiota is more complex than expected. a large study was implemented in our laboratory over a threeyear period (january through december ) to perform an exhaustive etiologic diagnosis of pneumonia. the study involved three icus in the public hospitals of marseille, france (one medical icu and two medico-surgical icus). a total of bal fluids, blood samples and urinary samples from icu pneumonia patients were studied. a diagnosis of community-acquired pneumonia, ventilator-associated pneumonia and aspiration pneumonia was defined as previously described [ ] [ ] [ ] . bal and blood sampling were performed as previously described [ ] . a cohort of icu patients without pneumonia was studied as controls. pneumonia patients exhibited episodes of community-associated table . comparison of lung microbiota between different studies. harris et al. [ ] bittar et al. [ ] bahranimougeot et al. [ ] erb-downward et al. [ ] hilty et al. [ ] studied nucleic acid extraction, pcr amplification, cloning and sequencing bacterial and fungal dna extraction from bal samples was performed on a magnapure lc workstation (roche diagnostics, meylan, france), using a magna pure lc dna isolation kit ii (roche diagnostics) as previously described [ ] . viral nucleic acids were extracted from ml of bal fluids using an mdx workstation and the qiaamp virus biorobot mdx kit according to the manufacturer's instructions. dna was tested by pcr for bacteria using broad-range primers targeting the s rdna gene; pcr was also used to test for universal fungi using broad-range primers targeting intergenic spacer of s rdna gene (eurogentec, seraing, belgium) ( table ). pcr product was cloned and approximately clones were screened per library. pcr, cloning and sequencing were performed as previously described [ ] . the obtained sequences were assembled and analyzed by chromaspro software and then blasted against those available in the genbank database (www.ncbi.nlm.nih.gov) for species identification. chimeric sequence search was performed with black box chimera check (b c ) program [ ] and by examining the blast profile of each sequence. suspected chimeric sequences were discarded from the study. sequences showing a similarity of . % were considered to be known species, whereas sequences showing a similarity of , % were considered to be novel species. legionella sp., afipia sp.,bradyrhizobium sp., azorhizobium sp., mesorhizobium sp., balneatrix alpaca and pneumocystis carinii were tested by pcr using specific primers followed by sequencing of pcr products ( table ). the sequences have been deposited in the genbank database (accession nu jf -jf ). standard bacteriological bal culture and blood culture as phenotypic identification of isolated bacteria were performed as previously described [ , ] . a cfu cut-off defined a positive bal culture. blood culture were processed as previously described [ ] . identification of fungi present in bal or blood samples was performed using a standard culture as previously described [ , ] . viral culture for cytomegalovirus, herpes simplex virus, parainfluenza viruses (types and ), respiratory syncytial virus, varicella-zoster virus, influenza viruses (type a and b), and enterovirus was performed using shell-vial culture as previously described [ , ] . amoeba co-culture were performed in microplates on acanthamoeba polyphaga as previously described [ ] . tentative isolations of mycobacterium sp., legionella sp. and mycoplasma pneumoniae were performed by using bactec mb automate, bcye agar plates and sp medium as previously described [ ] [ ] [ ] . results obtained using routine culture are available in table s ,s ,s ). mycobacterium sp., m. tuberculosis, m. avium group, bosea sp, parachlamydia sp., coxiella burnetii, chlamedia pneumoniae, chlamedia psittaci, mycoplasma pneumoniae, aspergillus sp., mimivirus, cmv, hsv, parainfluenza viruses and , respiratory syncytial virus, rhinovirus, metapneumovirus, varicella-zoster virus, influenza viruses a and b, enterovirus, and coronaviruses oc- , -e and nl- were detected using quantitative pcr. quantitative pcr was performed using a lightcyclerh instrument (roche diagnostics, meylan, france) in conjunction with the quantitect probe pcr kit. primers and probes used to identify these microorganisms are reported in table . the reaction was performed as previously described [ ] . for rna viruses, rna was first reverse transcribed using multiscribe tm reverse transcriptase (applied biosystems, courtaboeuf, france) as previously described [ ] . sera from patients were tested by immunofluorescent assay (ifa) for coxiella burnetii, bartonella quintana, bartonella henselae, legionella pneumophila, legionella anisa [ , , ] . viral serologies for adenovirus, cytomegalovirus, herpes simplex, parainfluenza viruses and , varicella-zoster virus and, influenza viruses a and b were performed using standard serologic methods (immunofluorescent assay or enzyme linked immunosorbent assay) [ ] . hemagglutination inhibition, immunoperoxidase staining and elisa techniques were used in-house to identify aspergillosis. l. pneumophila antigenuria and cmv pp antigenemia were tested for as previously reported [ , ] . results obtained using routine serology and antigenemia for viruses and fastidious pathogens are available in table s . bacterial and fungal nucleic acid sequences obtained from broad-range primer pcr were aligned with bioedit program (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and phylogenetic trees were create with mega software version . using the neighbor-joining method and the kimura- parameter [ ] . species having sequence similarities , % with those available in genbank databases were also blasted and classified in silico using ''classifier'' program in the ribosomal database project (http:// rdp.cme.msu.edu/) [ ] . statistical analyses were performed using chi square test, fisher's exact test, students t-test or mantel-haenszel's chi square test when appropriate. p values that were less than or equal to . were considered significant. the pubmed database (www.ncbi.nlm.nih.gov/pubmed/) and google website (http://www.google.fr/) were used to search whether species identified in our study had been previously reported in cases of pneumonia for articles published between and march , with the combined search term ''species name'' and ''pneumonia'', ''lung'' or ''infection.'' additional articles were identified by hand-searching the references of selected papers. additional search terms included ''microbiology'', ''diagnosis'', '' s'' and ''molecular detection'' were used. only publications in english were considered. papers in languages other than english were considered only when their abstracts in english were available. figure s schematic representation of microorganisms commonly identified in pneumonia and control cohorts, and those only detected in one cohort. fungi are shown in rectangles, viruses in octagons, and bacteria in circles. the name of each microorganism is indicated. (tif) figure s schematic representation of microorganisms that were commonly identified between each pneumonia form and controls, and those which were detected in only one cohort. fungi are shown in rectangles, viruses in octagons, and bacteria in circles. actinobacteria are shown in red, bacteroidetes in yellow, chlamydiae in orange, firmicutes in green, fusobacteria in purple, proteobacteria in blue and tenericutes in sky blue. cap, community-acquired pneumonia; vap, ventilatorassociated pneumonia; nv icu-p, non-ventilator icu pneumonia; ap, aspiration pneumonia; and cs, control subjects. (tif) figure s comparison of the bacterial communities found in our study with those found in lung specimens in three previous studies. novel phylotypes are not shown. actinobacteria are shown in red, bacteroidetes in yellow, chlamydiae in orange, firmicutes in green, fusobacteria in purple, proteobacteria in blue and tenericutes in sky blue. the name of the first author of each study and the name of each bacterium are indicated. the comparative analysis was conducted using cytoscape software. vap, ventilator-associated pneumonia; cf, cystic fibrosis. (tif) figure s molecular methods compared to standard routine culture for bacteria identification. text s bal culture, blood culture and serology results. data s detailed data about the relative abundance and richness of each species in their corresponding library. data s schematic data about the relative abundance and richness of each species in the corresponding library. table s species only detected in bal from pneumonia patients by molecular assays. (docx) nosocomial pneumonia in the intensive care unit acquired during mechanical ventilation or not etiology and diagnosis of pneumonia requiring icu admission guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients cytomegalovirus. an unexpected cause of ventilator-associated pneumonia viral infections in the icu herpes simplex virus: a marker of severity in bacterial ventilator-associated pneumonia bronchoscopic bal in the diagnosis of ventilatorassociated pneumonia molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis microbial diversity in the sputum of a cystic fibrosis patient studied with s rdna pyrosequencing molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients tropheryma whipplei in patients with pneumonia detection of plant dna in the bronchoalveolar lavage of patients with ventilator-associated pneumonia molecular analysis of oral and respiratory bacterial species associated with ventilator-associated pneumonia analysis of the lung microbiome in the ''healthy'' smoker and in copd disordered microbial communities in asthmatic airways chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients acetobacter indonesiensis pneumonia after lung transplant francisella philomiragia adenitis and pulmonary nodules in a child with chronic granulomatous disease severe pneumonia with leptotrichia sp. detected predominantly in bronchoalveolar lavage fluid by use of s rrna gene sequencing analysis human polymicrobial infections selection for staphylococcus aureus small-colony variants due to growth in the presence of pseudomonas aeruginosa burkholderia pseudomallei, b. thailandensis, and b. ambifaria produce -hydroxy- -alkylquinoline analogues with a methyl group at the position that is required for quorum-sensing regulation pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by staphylococcus epidermidis analysis of pseudomonas aeruginosa -hydroxy- -alkylquinolines (haqs) reveals a role for -hydroxy- -heptylquinoline in cell-to-cell communication prokaryote-eukaryote interactions identified by using caenorhabditis elegans candida albicans impairs macrophage function and facilitates pseudomonas aeruginosa pneumonia in rat molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections molecular and cultural analysis of the microflora associated with endodontic infections colonization of dental plaque: a source of nosocomial infections in intensive care unit patients genetic relationships between respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from patients in the intensive care unit undergoing mechanical ventilation infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults amoebaresisting bacteria and ventilator-associated pneumonia aspiration pneumonitis and aspiration pneumonia clinical significance of a positive serology for mimivirus in patients presenting a suspicion of ventilator-associated pneumonia black box chimera check (b c ): a windows-based software for batch depletion of chimeras from bacterial s rrna gene datasets direct identification of bacteria in positive blood culture bottles by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry evaluation of nested and real-time pcr assays in the diagnosis of candidaemia quantification of leishmania infantum dna by a real-time pcr assay with high sensitivity ameba-associated microorganisms and diagnosis of nosocomial pneumonia isolation of new fastidious alpha proteobacteria and afipia felis from hospital water supplies by direct plating and amoebal co-culture procedures cost-effectiveness of blood agar for isolation of mycobacteria diagnosis of legionnaires' disease. an update of laboratory methods with new emphasis on isolation by culture mycoplasma and ureaplasma. - q fever serology: cutoff determination for microimmunofluorescence value of microimmunofluorescence for diagnosis and follow-up of bartonella endocarditis detection of legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay mega : molecular evolutionary genetics analysis (mega) software version . naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy key: cord- -nc a r authors: kuczia, pawel; zuk, joanna; iwaniec, teresa; soja, jerzy; dropinski, jerzy; malesa-wlodzik, marta; zareba, lech; bazan, jan g.; undas, anetta; bazan-socha, stanislawa title: citrullinated histone h , a marker of extracellular trap formation, is increased in blood of stable asthma patients date: - - journal: clin transl allergy doi: . /s - - - sha: doc_id: cord_uid: nc a r background: emerging data indicates that extracellular traps (ets), structures formed by various immune cell types, may contribute to the pathology of noninfectious inflammatory diseases. histone hypercitrullination is an important step in ets formation and citrullinated histone h (h cit) is considered a novel and specific biomarker of that process. in the present study we have evaluated circulating h cit in stable asthmatics and investigated its relationship with asthma severity, pulmonary function and selected blood and bronchoalveolar lavage (bal) biomarkers. methods: in white adult stable asthmatics and well-matched controls we measured serum levels of h cit. in asthmatics we also performed bronchoscopy with bal. we analyzed blood and bal biomarkers, including interleukin (il)- , il- , il- , il- , il- p , il- a and interferon γ. for statistical analysis, mann–whitney u-test, χ( ) test, one-way ancova, roc curve analysis and univariate linear regression were applied. independent determinants of h cit were established in a multiple linear regression model. results: asthma was characterized by elevated circulating h cit ( . [ . – . ] vs. . [ . – . ] ng/ml, p = . ). in asthmatics positive associations were demonstrated between serum h cit and lung function variables, including total lung capacity (tlc) (β = . [ % ci . – . ]) and residual volume (β = . [ % ci . – . ]). h cit was increased in asthma patients receiving systemic steroids (p = . ), as well as in subjects with bal eosinophilia above cells/ml (p = . ). in asthmatics, but not in controls, circulating h cit correlated well with number of neutrophils (β = . [ % ci . – . ]) and monocytes (β = . [ % ci . – . ]) in peripheral blood. furthermore, bal macrophages, bal neutrophils, tlc, high-sensitivity c-reactive protein, il- p and bronchial obstruction degree were independent determinants of h cit in a multivariate linear regression model. conclusions: asthma is characterized by increased circulating h cit likely related to the enhanced lung ets formation. inhibition of ets might be a therapeutic option in selected asthma phenotypes, such as neutrophilic asthma. asthma is a common chronic inflammatory disease of the airways with a complex pathomechanism involving diverse immune processes [ , ] accompanied by a low-grade persistent systemic inflammation [ ] . emerging data indicate that extracellular traps (ets) may contribute to the pathology of noninfectious inflammatory diseases [ ] [ ] [ ] . ets are web-like structures coated with histones, granular and cytosolic proteins, formed by various immune cells, including neutrophils, eosinophils, and macrophages [ ] . neutrophil ets (nets) were first discovered as being mainly implicated in the innate immune response in host defense, enabling neutrophils to immobilize and kill invading bacteria, fungi or even viruses [ , ] . the impact and presence of ets formation in asthma have not been extensively studied yet, however, previous studies suggest that nets [ , ] and eosinophil extracellular traps (eets) [ , ] may contribute to the persistent airway inflammation in asthma. moreover, toussaint et al. [ ] have shown that rhinovirus respiratory tract infection, the most common cause of asthma exacerbation in humans, induces host-derived double strand dna release in nasal lavage samples of patients with mildmoderate asthma. there is also a growing body of evidence that ets may be entangled in clotting of the blood. we have recently reported evidence of a prothrombotic state in asthma which is characterized by enhanced plasma thrombin formation, impaired clot lysis and platelet activation [ ] , all of them related to the low-grade systemic inflammation [ ] , endothelial injury [ ] , elevated exacerbation rate [ ] , and likely increased atherosclerotic risk [ , ] . higher risk of thromboembolic events in asthmatics has been also demonstrated in epidemiological studies [ ] [ ] [ ] [ ] [ ] . the mechanisms functionally underlying this phenomenon are currently under investigation; however, ets contribution may be of importance. one of the key steps in ets formation is citrullination of histones performed by the histone-specific enzyme peptidylarginine deiminase (pad ) [ ] . emerging data indicates that citrullinated histone h (h cit), a key component of ets, may be recognized as a specific marker of ets formation both in tissue samples [ ] [ ] [ ] and in peripheral blood [ ] [ ] [ ] [ ] . taking into account low-grade systemic inflammation demonstrated in asthma and important role of ets formation in human pathology, we sought to evaluate serum h cit, a specific biomarker of ets formation, in asthma. we also studied its relation to asthma severity, lung function abnormalities, and selected blood and bronchoalveolar lavage asthma biomarkers. the study was conducted in the department of internal medicine, jagiellonian university medical college, krakow, poland, from june to january . we enrolled white adult patients with clinically stable asthma according to the global initiative for asthma (gina) guidelines [ ] and well-matched controls. the study participants were - years old. diagnosis of asthma was established based on a history of recurrent respiratory symptoms (wheeze, cough, shortness of breath, and chest tightness) together with current and/or historically documented postbronchodilator increase in forced expiratory volume in one second (fev ) of % and at least ml from the baseline [ ] . atopic status was confirmed by a positive skin prick testing for at least one inhaled allergen (allergopharma, reinbeck, germany). all asthma medications, except for biological therapy, were permitted, including oral corticosteroids at a daily dose equivalent to ≤ mg of prednisolone, only if the dose was unchanged for consecutive months. asthma patients were eligible if they had no exacerbation during the preceding months. severity of asthma was categorized according to the gina guidelines [ ] . "mild" asthma was defined as a mild persistent disease, treated with short acting β -agonist on demand, with or without low daily dose of inhaled corticosteroids (ics) (< μg of fluticasone propionate [fp] [dry powder inhaler] or equivalent). "moderate" asthma was defined as a mild persistent disease treated with a low (combined with long-acting β -agonists) or medium dose of ics ( - μg of fp or equivalent). "severe" asthma was defined as severe persistent disease despite using high daily dose of ics (> μg of fp or equivalent). asthma symptom control was assessed based on result of asthma control test (act). scores - were classified as "well-controlled asthma", - as "not well-controlled", while - as "very poorly controlled asthma". spirometry and bronchial reversibility test with μg of albuterol as well as postbronchodilator body plethysmography with assessment of residual volume (rv) and total lung capacity (tlc) were measured in all enrolled asthma patients according to the american thoracic society (ats) standards [ ] , using a jaeger masterlab spirometer (jaeger-toennies gmbh; hochberg, germany). in asthma patients we also performed bronchoscopy with bronchoalveolar lavage (bal). exclusion criteria comprised any acute illness, congestive heart failure, atrial fibrillation, coronary heart disease, hyper-or hypothyroidism, liver injury, chronic kidney disease (stage or more), autoimmune disease, malignant neoplasm or medical history of thromboembolism. in turn, arterial hypertension, diabetes mellitus, or hypercholesterolemia were allowed as comorbidities in subjects studied. arterial hypertension was defined based on a history of hypertension (blood pressure > / mmhg), or present antihypertensive treatment. diabetes mellitus was defined as the current use of insulin, or oral hypoglycemic medications, or fasting serum glucose > . mmol/l. hypercholesterolemia was defined as serum total cholesterol > . mmol/l or previous diagnosis along with lipid-lowering treatment. ex-smokers were eligible for enrolment if they had stopped smoking at least years before inclusion. control subjects were enrolled from the hospital personnel and its relatives. they were matched with patients according to age, sex, bmi, smoking status and internal medicine comorbidities. subjects with history of allergic diseases or bronchial obstruction were excluded. each control was individually matched with two patients considering the closest values of the matching criteria. bronchofiberoscopy was performed in asthma subjects according to the ats guidelines [ ] using the bf t bronchoscope (olympus, usa) with local anesthesia ( % lidocaine) and mild sedation ( . - . mg fentanyl and . - mg midazolam, both intravenously). bal was performed with ml of normal saline applied into the right middle lobe bronchus. first ml aliquot of obtained bal fluid was discarded, while the next sample was used for investigation. the cytospin preparations (thermo scientific, walthman, ma) were made from bal fluid samples and stained with the may-grunwald giemsa dye. percentages of inflammatory cells among cells in each preparation were counted (with exception of epithelial cells). remaining bal fluid was centrifuged ×g for min at room temperature, supernatant was frozen in aliquots and stored at − °c until analysis. fasting blood samples were drawn from the antecubital vein between : and : a.m, using minimal stasis. lipid profile, glucose, creatinine, urea, alanine aminotransferase, as well as complete blood cell and platelet count were assayed by routine laboratory techniques. fibrinogen was determined with the clauss method. high-sensitivity c-reactive protein (hscrp) and immunoglobulin e (ige) were measured by latex nephelometry (siemens, marburg, germany). blood samples were drawn into serum separation tube, centrifuged ×g for min, at room temperature. the supernatant was frozen in aliquots and stored at − °c until analysis. high sensitivity immunoenzymatic assays were used to measure levels of interleukin(il)- , il- , il- , il- , il- p , il- a, and interferon (inf)γ (ebiosciencea, vienna, austria, all) in serum and bal fluid of asthmatics and in serum of ( %) controls. concentration of h cit in serum was measured using elisa kit developed by cayman chemicals (ann arbor, mi, usa). this assay employed a monoclonal antibody specific for histone h citrullinated at r , r , and r (clone d ). the lower limit detection of the assay was . ng/ml, the upper ng/ml. analyses were carried out using statistica software package version . (tibco inc). the shapiro-wilk test has shown that continuous variables were non-normally distributed. they were reported here as median and interquartile range and compared using the mann-whitney u-test. categorical variables were given as percentages and compared by χ test with yates' correction, if applicable. age, sex, and body mass index (bmi) were considered as potential confounders for laboratory investigations. therefore, the box-cox normality transformation was used and a one way covariance analysis (ancova) was performed to adjust for confounding factors. to test for associations between two continuous variables univariate linear regression model was applied with adjustment for sex, age, and bmi. independent determinants of h cit were established in a multiple linear regression model, built by a forward stepwise selection procedure, verified by f snedecor's statistics, with f > . the r was used as a measure of the variance. cut-off points of bal and blood biomarkers in relation to circulating h cit levels were calculated in asthmatics based on receiver operating characteristic (roc) curves. moreover, to compare biomarkers between h cit-high and h cit-low asthma subjects the th percentile value of the circulating h cit in asthma individuals has been taken into account. in each case of multiple comparisons bonferroni correction has been applied and the nominal level of significance has been reduced proportional to the total number of all tests performed in multiple comparisons procedure. results were considered significant when the p value was less than . . clinical and laboratory characteristics of subjects studied are given in tables and , respectively. among asthmatics ( . %) subjects had atopy, while ( %) were characterized by severe disease according to gina [ ] . severe asthma was an indication for the use of systemic corticosteroids in patients ( . %). asthma individuals were characterized by increased serum levels of il- , il- (fig. b, c) , alanine aminotransferase activity, as well as elevated monocyte, eosinophil, lymphocyte, and total white blood cell (wbc) counts as compared to controls ( table ) . results of laboratory bal investigations are presented in table . as it has been demonstrated, il- and il- p were above the detection threshold in the majority of asthma patients. in turn, concentrations of il- , il- , il- a, and ifnγ in bal and blood, as well as il- in bal were below the detection threshold in the majority of subjects (additional file : tables s and s ). as expected, in asthma concentration of il- in bal was associated with bal and blood neutrophilia (β = . [ % ci . - . ] and β = . [ % ci . - . ], respectively). circulating h cit was significantly elevated in asthmatics in comparison to control group ( . [ . - . ] vs. . [ . - . ] ng/ml, p = . ), also after adjustment for potential confounders (p = . ) (fig. a) [ . - . ] ng/ ml, p = . ). administration of other drugs, as well as comorbidities had no impact on that parameter. in asthma, but not in controls, circulating h cit remained in positive associations with number of monocytes (β = . [ % ci . - . ]) and neutrophils [ . - . ] ng/ml vs. . [ . - . ] ng/ml, p = . ), as well as with bal il- levels higher than . pg/ml ( . [ . - . ] ng/ ml vs. . [ . - . ] ng/ml, p = . ) (additional file : fig. s ). interestingly, in controls h cit was directly associated with serum il- (β = . [ % ci . - . ]). in asthmatics, serum h cit was strongly related to the results of lung function tests, including tlc (β = . [ % ci . - . ]) and rv (β = . [ % ci . to . ]), also after adjustment for potential confounders. asthma patients with higher h cit, defined as values above . ng/ml ( th percentile as the cut-off point) had higher tlc in comparison to the remainders ( . [ . - . ] vs. . [ . - . ] ; p = . ) (additional file : fig. s c ). moreover, a weak negative relationship independent determinants of circulating h cit in asthmatics were identified using forward stepwise selection procedure. the number of macrophages and neutrophils in bal, hscrp, tlc, and the degree of prebronchodilator airway obstruction were demonstrated as positive predictors of h cit level in peripheral blood. in turn, surprisingly, il- (p ) had a negative impact on circulating h cit in that analysis. all aforementioned variables explained % of h cit variability (table ). in the present study we have demonstrated that serum h cit, a novel biomarker of ets formation, is increased in stable asthma subjects. although serum h cit was not related to the asthma severity according to the gina guidelines, a weak negative correlation with fev /vc suggests that enhanced bronchial obstruction, characteristic for more severe asthma subtype, might be associated with ets release. the same applies to the tendency towards elevated circulating h cit in those on systemic steroids or even anti-leukotriene medications. to our knowledge there is no evidence for upregulation of ets formation by steroids [ ] or leukotriene antagonists, however we cannot exclude such a relationship. previously, dworski et al. [ ] have found evidence for lung eets and nets formation in endobronchial biopsy specimens of mild and well-controlled asthma patients. in our study, the positive correlations demonstrated for tlc or rv and circulating h cit suggest that indeed, lungs might be the site of ongoing ets formation with subsequent h cit release into the peripheral blood of stable asthmatics. it might be assumed that in asthma higher lung volume and capacity, thus larger inflammatory site area, might result in the increase of local ets generation. interestingly, in asthma serum h cit was related to the number of monocytes and neutrophils in peripheral blood, as well as macrophages in bal. it is an intriguing result, as the issue of extracellular trap formation by macrophages remains a subject of ongoing research and debate [ ] . on the other hand, we found no direct association between serum h cit and number of neutrophils in bal. that finding might be to some extent explained by the relatively low percentage of patients with neutrophilic bal in our cohort, as other studies demonstrated increased nets in neutrophilic asthma sputum [ ] . however, we found increased h cit among asthmatics with higher bal eosinophilia. previously, choi et al. [ ] have demonstrated likely impact of eosinophils' derived eets on airway inflammation. our results stay in line with their finding, although bal composition does not necessarily reflect lung tissue inflammation [ ] . associations between circulating h cit and blood monocytes or neutrophils were not directly documented in the multivariate linear regression model, as hscrp, a biomarker closely related to monocyte and neutrophil counts in our study (data not shown), was a more precise determinant of serum h cit. moreover, in that model bal macrophages and bal neutrophils remained independent h cit determinants, suggesting the role of h cit as a potential biomarker of neutrophilic asthma even despite the lack of a direct correlation between bal neutrophils and h cit. interestingly, in our asthma cohort higher concentrations of bal il- was associated with increased circulating h cit. in turn, elevated serum il- in asthmatics stays in line with the previous reports of persistent low-grade systemic inflammation in asthma. links between nets and cytokine release have already been documented, e.g. nets may induce the transcription of il- and pro-il- β genes in macrophages in early atherosclerosis [ ] . surprisingly, in our study asthma patients were characterized by il- elevation as compared to controls. il- is rather known for its anti-inflammatory properties including inhibition of nets release [ ] . moreover, borish et al. have found diminished il- bal fluid concentrations in asthmatics [ ] . however, the observed increase in serum il- in our study together with the negative correlation between bmi and h cit in asthma subjects make further research of interactions between cytokines, adiponectin and ets release of interest. interestingly, serum h cit was negatively determined by serum il- (p ), a major player in the initiation and maintenance of t helper cells response [ ] . il- is known to inhibit airway inflammation and bronchial obstruction in murine models of allergic asthma [ ] . an interventional study with recombinant il- in human asthmatics showed a decrease in peripheral blood and sputum eosinophilia with no impact on bronchial reactivity [ ] . another function of il- is the inhibition of pathological angiogenesis [ ] . it has been demonstrated that abnormal neovascularization is an important feature of airway remodeling in asthma patients [ , ] . we cannot exclude, that in asthmatics with enhanced ets formation decrease of il- might contribute to the unfavorable airway remodeling and bronchial neovascularization. notably, increased h cit in peripheral blood of asthma subjects is particularly important, since there is a growing body of evidence that enhanced ets formation might contribute to the development of various systemic sequelae, including thrombotic complications or autoimmunity [ ] [ ] . in particular, the presence of autoantibodies against citrullinated peptides in rheumatoid arthritis, another noninfectious inflammatory disease, is likely related to the pad activity enabling ets release [ , ] . in turn, severe asthma patients present more frequently with autoimmune diseases [ ] . thus, production of ets with subsequent release of related products into the circulation may provide the initial stimulus for autoimmunity, explaining the increased rate of autoimmune diseases in asthmatics. moreover, it has been demonstrated that histones might directly contribute to inflammatory injury by a host tissue damage [ ] [ ] [ ] [ ] , including microvascular endothelial barrier disruption and subsequent cell inflow into the inflammatory site [ , , ] . experimental approach with specific h cit inhibition led to improved outcomes in mouse sepsis models [ , ] . although this is only a hypothesis, we believe that even relatively low concentrations of h cit and other citrullinated histone types may contribute to the endothelium damage in asthmatics. the availability of pharmacologic histone inhibitors makes it a particularly intriguing issue, as their use might be of benefit at least in some asthma phenotypes. furthermore, the potential contribution of ets formation to the prothrombotic phenomenon in asthma also deserves a comment. fuchs et al. [ ] has reported that nets trigger the blood clotting through activation of platelets, recruitment of red blood cells and stimulation of fibrin deposition, being a potential link between immune response and thrombosis. in particular, ammollo et al. [ ] revealed that extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein c activation. recently, zuo et al. [ ] documented increased h cit and myeloperoxidase-dna (mpo-dna) in sera of subjects with coronavirus disease (covid- ), illness accompanied by clotting impairment and microvascular thrombosis. thus, we speculate that elevated circulating h cit may, at least to some extent, explain prothrombotic blood alterations and increased risk of thromboembolic complications demonstrated even in well-controlled asthma patients [ , [ ] [ ] [ ] . the limitations of our study include relatively small sample size. particularly, subgroup analysis should be interpreted with caution. we determined each variable at a single time point, thus we cannot exclude changes of the variables during time. we did not analyze ets formation in a functional assay. however, the laboratory techniques for directly determining the ets formation are difficult and very demanding from the technical point of view. applied here h cit elisa assay is simple, but relatively novel, thus might require further validation before being established as a reliable test for the extensive use in practice. we analyzed h cit in blood and not in airways, which are the main loci of asthma inflammatory response. we did not measure other cytokines potentially relevant for the lung and systemic inflammatory response, including il- β and il- , released after nlrp stimulation by nets [ ] . statistical associations reported here might not necessarily indicate cause-effect relationships. finally, a clinical relevance of increased serum h cit in relation to asthma course, disease progression, exacerbation risk, or vascular outcomes remains to be established. asthma is characterized by increased circulating h cit likely related to the enhanced lung ets formation. although large experimental and observational studies are needed to verify this hypothesis, it seems that ets release is involved in asthma pathogenesis. thus inhibition of ets might be a therapeutic option in selected asthma phenotypes, such as neutrophilic asthma. the immunology of asthma the relationship of airway structural changes to blood and bronchoalveolar lavage biomarkers, and lung function abnormalities in asthma prothrombotic state in asthma is related to increased levels of inflammatory cytokines, il- and tnfα biological function of eosinophil extracellular traps in patients with severe eosinophilic asthma netopathic inflammation in chronic obstructive pulmonary disease and severe asthma an emerging role for neutrophil extracellular traps in noninfectious disease to net or not to net:current opinions and state of the science regarding the formation of neutrophil extracellular traps neutrophil extracellular traps in immunity and disease neutrophil autophagy and extracellular dna traps contribute to airway inflammation in severe asthma neutrophil extracellular traps are associated with inflammation in chronic airway disease eosinophil and neutrophil extracellular dna traps in human allergic asthmatic airways host dna released by netosis promotes rhinovirus-induced type- allergic asthma exacerbation asthma is associated with enhanced thrombin formation and impaired fibrinolysis endothelial dysfunction and pentraxin- in clinically stable adult asthma patients impaired fibrinolysis and lower levels of plasma α -macroglobulin are associated with an increased risk of severe asthma exacerbations increased blood levels of cellular fibronectin in asthma: relation to the asthma severity, inflammation, and prothrombotic blood alterations increased activity of lipoprotein-associated phospholipase a in non-severe asthma risk of deep vein thrombosis and pulmonary embolism in asthma asthma increases pulmonary thromboembolism risk: a nationwide population cohort study risk of pulmonary embolism and deep venous thrombosis in patients with asthma: a nationwide case-control study from sweden relation of adult-onset asthma to coronary heart disease and stroke asthma predicts cardiovascular disease events netosis markers: quest for specific, objective, and quantitative markers eosinophils release extracellular dna traps in response to aspergillus fumigatus extracellular traps derived from macrophages, mast cells, eosinophils and neutrophils are generated in a time-dependent manner during atherothrombosis validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone h as a marker for neutrophil extracellular traps in human plasma predictors of neutrophil extracellular traps markers in type diabetes : • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over ready to submit your research ? choose bmc and benefit from: mellitus: associations with a prothrombotic state and hypofibrinolysis cith : a reliable blood biomarker for diagnosis and treatment of endotoxic shock cl-amidine prevents histone citrullination and neutrophil extracellular trap formation, and improves survival in a murine sepsis model global initiative for asthma. global strategy for asthma management and prevention recommendations for a standardized pulmonary function report an official american thoracic society technical statement position paper on guidelines for fiberoptic bronchoscopy in adults neutrophil extracellular traps are downregulated by glucocorticosteroids in lungs in an equine model of asthma macrophage extracellular traps: a scoping review interleukin- regulation in normal subjects and patients with asthma the p subunit of interleukin (il)- promotes stabilization and export of the p subunit: implications for improved il- cytokine production interleukin inhibits antigen-induced airway hyperresponsiveness, inflammation, and th cytokine expression in mice effects of recombinant human interleukin- on eosinophils, airway hyper-responsiveness, and the late asthmatic response new insights into il- -mediated tumor suppression bronchial angiogenesis in severe glucocorticoid-dependent asthma angiogenesis in paediatric airway disease pad : pathophysiology, current therapeutics and future perspective in rheumatoid arthritis the role of extracellular histone in organ injury extracellular histones are major mediators of death in sepsis impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation circulating histones are major mediators of cardiac injury in patients with sepsis citrullinated histone causes endothelial barrier dysfunction endothelial barrier dysfunction in septic shock citrullinated histone h -a novel target for treatment of septic shock extracellular dna traps promote thrombosis extracellular histones increase plasma thrombin generation by impairing thrombomodulin-dependent protein c activation neutrophil extracellular traps in covid- the three cytokines il- β, il- , and il- α share related but distinct secretory routes publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. supplementary information accompanies this paper at https ://doi. org/ . /s - - - .additional file . table s . cytokine concentrations in peripheral blood of asthma and control subjects (all tested cytokines). table s . bal cytokine concentrations in asthmatics (all tested cytokines).additional file . figure s . comparisons of asthma patient subgroups distinguished on the basis of roc curve analysis.additional file . figure s . comparisons of h cit-low vs. h cit-high asthma patients. asthma patients were subdivided into h cit-high and h cit-low groups using blood h cit value of the th percentile as the cut-off point ( . ng/ml). key: cord- -diwigcy authors: de schutter, iris; de wachter, elke; crokaert, françoise; verhaegen, jan; soetens, oriane; piérard, denis; malfroot, anne title: microbiology of bronchoalveolar lavage fluid in children with acute nonresponding or recurrent community-acquired pneumonia: identification of nontypeable haemophilus influenzae as a major pathogen date: - - journal: clin infect dis doi: . /cid/cir sha: doc_id: cord_uid: diwigcy background. precise etiologic diagnosis in pediatric community-acquired pneumonia (cap) remains challenging. methods. we conducted a retrospective study of cap etiology in groups of pediatric patients who underwent flexible bronchoscopy (fob) with bronchoalveolar lavage (bal); children with acute nonresponsive cap (nr-cap; n = ) or recurrent cap (rec-cap; n = ). procedural measures were taken to limit contamination risk and quantitative bacterial culture of bal fluid (significance cutoff point, ≥ ( ) colony-forming units/ml) was used. blood culture results, serological test results, nasopharyngeal secretion findings, and pleural fluid culture results were also assessed, where available. results. an infectious agent was detected in . % of cases. in . % of infections, aerobic bacteria were isolated, of which . %, . %, and . % were haemophilus influenzae, moraxella catarrhalis, and streptococcus pneumoniae, respectively. most ( . %) of the h. influenzae strains were nontypeable (nthi). h. influenzae was detected in . % of nr-cap cases and . % of rec-cap cases, whereas mycoplasma pneumoniae was the predominant pathogen in the nr-cap group (accounting for . % of cases) but not in the rec-cap group ( . %). viruses were found in . % of cases, with respiratory syncytial virus, parainfluenzaviruses, and influenzaviruses detected most frequently. mixed infections were found in . % of nr-cap cases and . % of rec-cap cases. conclusions. a variety of microorganisms were isolated with frequent mixed infection. nthi was one of the major pathogens found, especially in association with recurrent cap, possibly because of improved detection with the fob with bal procedure. this suggests that the burden of pediatric cap could be reduced by addressing nthi as a major causative pathogen. direct sampling from the lung and lower airways can only be achieved with invasive methods, such as transthoracic needle aspiration (tna) and puncture of pleural effusion [ ] . in adults, sputum samples are considered to be a valuable, noninvasive alternative for bacterial detection, but it is often not feasible to obtain a sputum sample from children. therefore, most pediatric studies are based on samples obtained from the upper respiratory tract (urt) and blood samples. however, the urt is frequently colonized by bacterial pathogens [ , ] , making it difficult to discriminate commensal organisms and acute pathogens. moreover, although blood is normally sterile and blood cultures are highly specific, the sensitivity of blood cultures in childhood cap is low ( %- %) [ , ] . serological tests for the detection of viruses and atypical microorganisms are of limited use in clinical practice because of the need for convalescent-phase serum samples. flexible bronchoscopy (fob) with bronchoalveolar lavage (bal) is an option if noninvasive samples from the lower respiratory tract (lrt) are impossible to obtain or for selected patients with nonresponse to antibiotic treatment [ , ] . bal fluid has the advantage of being suitable for multiple detection methods, including microscopy, aerobic culture, viral culture, and polymerase chain reaction (pcr). protected brushes cannot pass the pediatric flexible bronchoscope, but the risk of oropharyngeal contamination can be reduced considerably with careful technique [ ] . interpretation of bal results is more reliable with the use of quantitative bacterial culture [ , , , , ] . the reliability of fob plus bal used with these precautions has been demonstrated, with some authors reporting specificities of %- % for potential pathogens in relation to actual pneumonia [ , ] . moreover, fob plus bal has been proven to be safe, even in critically ill children [ , ] . in this retrospective study, we assessed the etiology of cap in groups of pediatric patients who underwent fob plus bal: children with acute nonresponsive cap and children with recurrent cap. among children, these cap features account for a significant proportion of physician and emergency department visits, hospitalizations, and antibiotic prescribing in the community. in addition to bal fluid culture, blood cultures, serological testing, culture and pcr results of nasopharyngeal aspirates of nasopharyngeal secretions (npa), and pleural fluid culture were assessed, where available. this was a retrospective analysis of pediatric patients who underwent fob plus bal from january through december . the study was approved by the ethics committee of universitair ziekenhuis brussel (uz brussels, belgium). patients selected for analysis had acute nonresponsive community-acquired (broncho)pneumonia (nr-cap group) or recurrent communityacquired (broncho)pneumonia (rec-cap group). nr-cap case patients were defined according to current pediatric cap guidelines [ ] as patients with persistent fever (. . °c) and persistent elevated infection parameters in the peripheral blood, associated in some cases with worsening consolidation visible on chest radiographs after at least h of antibiotic treatment. the rec-cap group had an episode of cap with a history of at least other cap episodes. exclusion criteria included the presence of severe and chronic conditions (such as cystic fibrosis, primary ciliary disease, immune deficiency, bronchopulmonary dysplasia, cardiopathy, neuromuscular diseases, asplenia, and tuberculosis) and nosocomial respiratory tract infection. retrospective analyses were conducted of demographic data, antibiotic use before fob plus bal, and the results of a broad microbiological investigation. fob and bal were performed according to european respiratory society recommendations [ ] . a pediatric flexible fiberoptic bronchoscope (bf c olympus) was inserted orally to avoid nasal contamination. aliquots of saline solution ( . %) were instilled and reaspirated in the diseased lobar or segmental bronchus (maximum volume, ml/kg body weight). the first bronchial lavage fraction was discarded. all subjects were sedated with intravenous midazolam, atropinesulphate, and tramadol hydrochloride; were transcutaneously monitored for oxygen saturation and heart rate; and received supplementary oxygen as needed during the procedure. blood cultures and serological testing were performed according to routine procedures when cap was diagnosed, before starting antibiotic treatment. npa was performed routinely in children , years of age with cap with use of a flexible sterile catheter (vaginal catheter ch. ; medinorm) introduced into a nostril up to the nasopharynx. at this level, saline solution ( ml; . %) was instilled and immediately reaspirated. pleural fluid was obtained from patients with a significant amount of pleural fluid for safe puncture, using routine disinfection and puncture methods. samples were processed using standard techniques and interpreted as listed in table . for haemophilus influenzae biotyping and capsular typing using a pcr and agglutination technique were performed at centre hospitalier universitaire st pierre, brussels. nontypeable h. influenzae (nthi) strains were distinguished from nonhemolytic haemophilus haemolyticus [ , ] . b-lactamase activity was assessed using a nitrocephin-based test, and antibiotic susceptibility was determined by the etest technique (biomérieux). serotyping and antibiotic susceptibility testing of streptococcus pneumoniae was performed as described elsewhere [ ] . viral culture and an in-house multiplex pcr (mpcr) were used on bal fluid and npa to detect respiratory viruses and atypical microorganisms (mycoplasma pneumoniae and chlamydophila pneumoniae). serological tests were performed by the complement fixation technique. the v test with yate's correction or, when one of the subjected figures was , , the fisher's exact test were used to compare positivity rates between the groups. statistically significant differences were defined as those for which the probability of occurrence was % %. of the children who had fob plus bal, children- with nr-cap and with rec-cap-met the inclusion criteria. the median age and sex ratio in both groups were similar ( table ). most patients with nr-cap ( %) had received antibiotic treatment within h before fob plus bal, whereas most patients with rec-cap ( %) had not been treated ( table ) . a pathogen was detected in approximately three-quarters of children in both groups (table ) . exclusively bacterial pathogens were found in . % of the nr-cap group and . % of the rec-cap group, and mixed infection was found in . % and . %, respectively. all bacterial pathogens except s. pneumoniae were isolated from bal specimens, whereas only blood cultures and pleural fluid cultures were positive. in both patients with positive blood cultures, s. pneumoniae was isolated from the blood cultures, whereas h. influenzae and m. catharralis were isolated from bal specimens from the same patients. in patient, staphylococcus aureus was isolated from pleural fluid culture, whereas bal yielded the same organism in combination with enterobacter aerogenes. in the latter case, s. pneumoniae was isolated from the pleural fluid specimen, but bal and blood cultures remained negative. overall, aerobic bacteria were isolated from ( . %) of patients with infection; . % of the aerobic bacteria isolated were h. influenzae. bacterial pathogens were found significantly more often in patients with rec-cap than in patients with nr-cap ( (table ). in the remaining cases, h. influenzae was found with another bacterial pathogen (moraxella catarrhalis in cases, s. pneumoniae in cases, and s. aureus in cases). h. influenzae was found in mixed bacterial-viral infections, mixed bacterial-atypical infections, and mixed infections with mycoplasma species and a respiratory virus (table ) . of the h. influenzae isolates, ( . %) were nthi, and were lost for typing. three isolates were initially misidentified by agglutination technique as being encapsulated strains, type e and type b. however, pcr results were negative in all cases, resulting in a definitive identification as nthi. the majority of h. influenzae isolates ( . %) were b-lactamase negative, including of isolates from nr-cap cases and of isolates from rec-cap cases. two b-lactamase-negative isolates were intermediate susceptible to ampicillin (minimum inhibitory concentration [mic], mg/l), but no b-lactamasenegative, fully ampicillin-resistant strain was detected. after h. influenzae, m. catarrhalis and s. pneumoniae were the next most common bacterial pathogens, accounting for . % and . % of cases, respectively, in which aerobic bacteria were isolated. m. catarrhalis was isolated in . % of nr-cap cases and . % of rec-cap cases, whereas s. pneumoniae was isolated in . % of nr-cap cases and . % of rec-cap cases ( table ). although s. pneumoniae was isolated in only cases, a variety of pneumococcal serogroups-serotypes (sgt) was found, with a predominance of sgt , , and , which were isolated in , , and cases, respectively. all s. pneumoniae isolates were fully susceptible to parenterally administrated penicillin (mic, % lg/ml), whereas isolates were intermediate susceptible to orally administrated penicillin (mic, . - lg/ml). m. catarrhalis and s. pneumoniae were mostly found with another bacterial pathogen in ( . %) of and ( . %) of cases, respectively ( atypical microorganisms alone were detected significantly more often in patients with nr-cap than in patients with rec-cap (table ) . m. pneumoniae was detected in . % of the samples from patients with nr-cap that were tested and in . % of the samples from patients with rec-cap that were tested, and c. pneumoniae was detected in . % of rec-cap cases (table ). viruses were found in . % of cases overall. exclusive viral infection was reported in . % of nr-cap cases and . % of rec-cap cases (table ) ; overall, respiratory syncytial virus (rsv), parainfluenzaviruses, influenzaviruses, adenovirus, and human metapneumovirus were detected most frequently (table ). viral coinfection was found in ( . %) of nr-cap and ( . %) of rec-cap cases. we assessed otherwise healthy children with either nr-cap or rec-cap to determine the etiology of the infection. a heterogeneous group of patients was included, which reflected pediatric cap cases that often require hospitalization in developed countries. an infectious agent was detected in % of cases, compared with detection in %- % of cases in previous studies of pediatric cap [ , , , ] . however, most of these studies involved urt samples, and thus had limited specificity for detecting bacterial pathogens, with few studies involving samples of the lrt or lung aspirates [ , , ] . among the aerobic bacteria, which were isolated in approximately half of infections, we found a predominance of nthi in both groups of children; approximately three-quarters were h. influenzae, of which . % were nthi. to our knowledge, this is the first such reported finding since those of shann et al [ ] and weinberg et al [ ] in the s. since then, studies involving children with cap or lrt infection (using serological testing or tna) have suggested a possible role for nthi in pediatric pneumonia but have failed to define it as a significant pathogen [ ] . for example, in a recent study involving children with cap, cases were attributed to s. pneumoniae and no case of h. influenzae infection was found using tna [ ] . however, the study included a small number of patients and investigated lobar cap only. it is also possible that nthi infection was missed in previous studies of pediatric cap because the methodology was not appropriate for nthi detection. nthi does not easily invade the bloodstream or pleura [ , ] , and misclassification of nthi strains as hib strains using the slide agglutination technique has been reported [ ] . also, in many tna studies, the culture media used were not appropriate for detecting h. influenzae and, if h. influenzae was found, strains were not serotyped [ ] . the reliability of our finding that nthi is frequently involved as a pathogen in childhood cap is supported by the sterility of the lrt in healthy subjects and the finding that, in children with unilateral cap, bal culture of the contralateral side has negative results [ , ] , as well as by the established pathogenicity of nthi in another normally sterile site, the middle ear. nthi is associated with persistent, nonresponsive acute otitis media (aom) [ , ] , possibly attributable to the choice of treatment (amoxicillin is used as first-line treatment), leading to the selection of b-lactamase-positive h. influenzae [ ] . this was not the case in our study, because % of nthi isolates were b-lactamase negative. in aom, nthi has a clear association with recurrence . month after the completion of antibiotic therapy, with an incidence of % in third and subsequent episodes [ ] . nthi infection contributes to complications in aom and is clinically indistinguishable from aom caused by s. pneumoniae [ ] . it is therefore possible chlamydophila pneumoniae / tested ( ) / tested ( . ) ns note. nr-cap, nonresponding community-acquired pneumonia; rec-cap, recurrent community-acquired pneumonia; coinfection: cases in which at least infective agents of the same microbiological group were isolated; ns, not significant. a n for viruses. that, as in aom, nthi is not an innocent bystander in the lrt. indeed, it is now increasingly recognized that nthi is capable of causing invasive disease, particularly neonatal sepsis, and is also a cause of cap, bacteremia and, to a lesser extent, meningitis [ , ] . moreover, we are confident that the risk of contamination in bal samples was limited considerably, because various technical measures were taken to avoid contamination by urt secretions and a significance cutoff value was used for bacterial bal culture [ , , [ ] [ ] [ ] . although a significance cutoff of r colonyforming units (cfu)/ml has been shown to be reliable in adults [ , ] , we have chosen to use a higher cutoff value of r cfu/ml, because colonization of the urt is more common in children. the finding that only . % of the haemophilus strains detected in our study were nonhemolytic h. haemolyticus, whereas previous studies showed that the prevalence of nonhemolytic h. haemolyticus in throat swab samples obtained from healthy children or adults was %- % [ , ] , also supports the reliability of our results. other typical bacterial pathogens for pneumonia, such as m. catarrhalis and s. pneumoniae, were detected in . % and . % of cases, respectively, in which aerobic bacteria were isolated. bacterial coinfections were reported in several earlier studies, including studies that used tna [ , , ] ; most such coinfections were due to s. pneumoniae and h. influenzae, although coinfections due to h. influenzae and m. catarrhalis, as recorded in our study, were also reported [ ] . although the role of each isolated pathogen cannot be defined, previous findings of coinfection and recent insights into interspecies quorum signaling suggest an individual role for each pathogen and/or possible interaction between them [ ] . in belgium, universal childhood vaccination against h. influenzae type b (hib) was introduced in the s and might explain the absence of any case of infection due to hib in our study. however, because universal childhood pneumococcal vaccination using pcv was only started in , it is very unlikely that it may have influenced our results. the proportion of cases with an identified etiology was comparable between both groups. bacterial etiology was found more often in rec-cap cases than in nr-cap cases, probably because only approximately a quarter of patients with rec-cap received antibiotics before fob plus bal [ ] . in antibiotic-pretreated patients, the diagnostic yield might have been increased with the use of bacterial serological testing [ ] or quantitative pcr [ ] . the relatively low detection rates for m. pneumoniae and c. pneumoniae may be related to the young age of the patients, because atypical infections are most common in children . years of age [ , , ] . m. pneumoniae was a predominant pathogen in the nr-cap group but not in the rec-cap group. most of the patients with nr-cap had been treated with penicillin or aminopenicillins, the first-line antibiotics for treatment of cap, and the predominance of m. pneumoniae gives a possible reason for nonresponsiveness. viruses were found in . % of cases, with rsv, parainfluenzaviruses, and influenzaviruses being detected most frequently. this incidence is relatively low, compared with the findings of other studies [ , ] , and may have been due to omission of rhinovirus in the pcr assay. virus detection was slightly more frequent in the nr-cap group. viruses were the sole pathogen more than twice as often in the nr-cap group than in the rec-cap group, possibly providing another explanation for nonresponsiveness to initial antibiotic therapy. viral coinfections, which were found in . % of cases overall, are rarely reported in most studies of cap, although one study reported an incidence of % for all cap cases and % for viral cap cases [ ] . mixed infections were found in . % of cases, which was comparable to previous findings [ , , ] . our findings are compatible with current guidelines that recommend antibiotic treatment for all cap cases, because an established viral or atypical etiology does not rule out a concomitant bacterial infection [ , ] . the predominance of nthi in rec-cap cases in our study suggests the use of aminopenicillins with or without b-lactamase inhibitors for the initial treatment in recurrent cases. in case of nonresponsiveness, the addition of macrolides to the initial treatment regimen is supported by the relatively high incidence of m. pneumoniae infection in nr-cap cases. in conclusion, as in previous studies, we found that a variety of microorganisms were involved in pediatric nonresponsive and recurrent cap with frequent mixed infection. in contrast to other reports, we found nthi to be a major bacterial pathogen, possibly because of improved detection with fob plus bal. this finding might explain cases of nonresponsiveness to antibiotic treatment or recurrence in pediatric cap, similar to observations in aom, which is frequently associated with nthi infection. additional research is needed to elucidate the pathogenic mechanisms of nthi in pediatric cap. however, our findings suggest that the burden of pediatric cap could be reduced by addressing nthi as a major causative pathogen in treatment and prevention strategies. the role of bronchoalveolar lavage in diagnosing nonopportunistic bacterial pneumonia 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we thank dr. joanne knowles, for her outstanding editorial assistance, and dr. jos bellens, of the department of medical informatics, universitair ziekenhuis brussel (uz brussel), for his help with database production.financial support. glaxosmithkline biologicals provided an unrestricted scientific grant.potential conflicts of interest. a. m. has participated in advisory boards on pneumococcal diseases and vaccines for wyeth-pfizer and glax-osmithkline; is a member of a steering committee of a project sponsored by wyeth-pfizer; received payment for the development of educational presentations from glaxosmithkline; is coordinating a steering committee of a research project sponsored by wyeth-pfizer; has been a speaker for wyeth-pfizer and/or glaxosmithkline on pneumococcal diseases and epidemiology; and has received travel expenses from glaxosmithkline, novartis, abbott, sanofi-pasteur, and pfizer (weyth). i. d. s. has participated in advisory boards on nthi for glaxosmithkline and advisory boards on pneumococcal diseases and vaccines for wyeth-pfizer and glaxosmithkline; is a member of a steering committee of a project sponsored by wyeth-pfizer; and has received travel expenses from glaxosmithkline and sanofi-pasteur. j. v. is a member of a steering committee of a research project sponsored by wyeth-pfizer and is heading the national reference laboratory for s. pneumoniae that received grants from wyeth-pfizer and glaxosmithkline for serotyping isolates of ipd studies. d. p. has received grants from janssen-cilag, astrazeneca, wyeth, and pfizer and travel expenses from pfizer.all other authors: no reported conflicts. 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 in the acknowledgements section. key: cord- - is rv c authors: piñana, josé luis; giménez, estela; gómez, maría dolores; pérez, ariadna; gonzález, eva maría; vinuesa, víctor; hernández-boluda, juan carlos; montoro, juan; salavert, miguel; tormo, mar; amat, paula; moles, paula; carretero, carlos; balaguer-roselló, aitana; sanz, jaime; sanz, guillermo; solano, carlos; navarro, david title: pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: is rv c objectives: to date no definitive cut-off value for cytomegalovirus (cmv) dna load in bronchoalveolar lavage (bal) fluid specimens has been established to discriminate between cmv pneumonia and pulmonary cmv dna shedding in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. methods: the current retrospective study is aimed at assessing the range of cmv dna loads quantified in bal fluid specimens from allo-hsct patients with pneumonia in which different microorganisms were causally involved. results: a total of bal specimens from patients were included. cmv dna was detected in out of bal fluid specimens and the median cmv dna load from patients in whom cmv pneumonia was unlikely or could be tentatively ruled out was ( – , ) iu/ml. the frequency of cmv dna detection and median cmv dna loads were comparable, irrespective of the attributable cause of pneumonia. detection of cmv dna loads in bal fluid specimens > iu/ml was independently associated with pneumonia-attributable mortality. conclusions: the current study highlights the difficulty in establishing universal cmv dna load thresholds in bal fluid specimens for distinguishing between cmv pneumonia and pulmonary cmv dna shedding, and suggests that the presence of cmv dna in bal fluid specimens beyond a certain level may have a deleterious impact on patient outcome. the implementation of effective prevention strategies has significantly reduced the incidence of cytomegalovirus (cmv) pneumonia after allogeneic hematopoietic stem transplantation (allo-hsct) ; nevertheless, this clinical entity persists as a major clinical problem owing to poor survival, despite timely initiation of targeted antiviral therapy. , currently, diagnosis of cmv pneumonia still remains a challenge. proven cmv pneumonia can only be diagnosed using virological, histopathological or immunochemistry methods on biopsied lung tissue ; this specimen type, however, is rarely obtained due to potential life-threatening complications. traditionally, culture-based bronchoalveolar lavage (bal) testing has been the mainstay for suggesting cmv involvement ex vivo , although detection of viable cmv in bal fluids only points to a probable causality. may be present in the absence of probable or proven disease (pulmonary cmv shedding). [ ] [ ] [ ] [ ] recent studies suggested that quantitation of cmv dna in bal fluids may permit discrimination between these two conditions - ; specifically, a cut-off cmv dna level > iu/ml was proposed to serve that purpose, this displaying a positive predictive value of roughly % for probable cmv pneumonia using current prevalence rates. nevertheless, validation of diagnostic viral load threshold across centers using different real-time pcr assays for cmv dna quantitation and procedures for bal obtention seems of paramount relevance. this task is hampered by the very low incidence of cmv pneumonia nowadays ( < %) , and the difficulty in establishing an incontrovertible diagnosis, even when lung tissue specimens are available for testing. - exploratory studies gathering information on the range of cmv dna loads measured in bal specimens from allo-hsct patients with pneumonia in whom cmv causality is unlikely or can be reasonably ruled out may provide useful information and are thus warranted. here, we report on our experience on this matter. the study cohort consisted of consecutive patients who received an allo-hsct at hospital clínico universitario-hcu-( n = ) or at hospital universitario politécnico "la fe" -hlf-( n = ) and underwent diagnostic bronchoscopy. clinical and microbiological data of patients and bal fluid specimens ( n = ; hcu, n = ; hlf, n = ) submitted to the respective microbiology service between may and may , respectively, were retrospectively reviewed. as per protocol, quantitative cmv pcr testing has been routinely performed on bal specimens at both centers since . all patients had clinical and radiography signs of pneumonia at the time of sampling. bronchoscopy was performed using standard procedures according to international consensus guidelines. the first bal fluid specimen per pneumonia event was taken into consideration for analysis purposes. this study was approved by the hospital clínico, fundación incliva ethics committee and informed consent was obtained from all patients. a preemptive antiviral therapy approach was used at hcu to prevent cmv end-organ disease. cmv dna in plasma was quantified using the realti me cmv pcr assay (abbott molecular, des plaines, il, usa), which exhibits a limit of detection of approximately iu/ml. patients were preemptively treated with oral valganciclovir, i.v. ganciclovir or i.v. foscarnet upon detection of cmv dna levels exceeding iu/ml or a cmv dna doubling time ≤ days, as previously reported. , surveillance for cmv dna detection and quantitation was conducted at least once a week within the first days after allo-hsct and this was extended beyond day in patients at risk for recurrent episodes of cmv dnaemia. in turn, a universal prophylaxis strategy was deployed at hlf. briefly, hla-matched related allo-hsct recipients were given oral valganciclovir ( mg/day, three times a week) through day after transplantation. unrelated allo-hsct recipients were treated with oral valganciclovir ( mg/day) through day after transplantation. at this center, plasma cmv dna load was quantified using the cmv r-gene® (biomerieux, l'etoile, paris, france), which displays a limit of detection of iu/ml, prior dna extraction using the virus/pathogens mini kit (qiagen, valencia, ca, usa) on the qiasymphony dsp platform (qiagen). plasma cmv dna monitoring was performed once a week within the first days, fortnightly from day to day , and every - weeks thereafter through day after transplantation. detection of any level of cmv dna in plasma prompted the administration of antiviral therapy with (val)ganciclovir or foscarnet at the doses specified above. anti-cmv therapy was given at the physician discretion when patients with cmv dna detected in bal specimens had concurrent cmv dnaemia with cmv dna levels below the threshold for preemptive antiviral therapy. plasma specimens obtained within h of bal sampling were available from all patients for cmv dna quantitation. all bal fluid specimens underwent cmv pcr testing within - h. upon reception. samples were kept at °c until processed. bal fluid specimens obtained from different locations (when available) were collected in sterile containers, pooled and vortexed for s after the addition of sterile glass beads. two-hundred μl of each undiluted bal fluid samples were subjected to nucleic acid extraction using the m sp system (abbott diagnostics) or the qiaamp dna blood mini kit (qiagen) on either the qia symphony or the ez- platforms (qiagen). cmv dna quantitation was performed using the realti me cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf. commutability of the cmv who international standard for cmv dna quantitation in bal specimens was assessed. the cmv who standard was reconstituted in ml of deionized water as recommended by the manufacturer. a pool of bal specimens free of cmv dna, as determined by the abbott pcr assay was made and spiked with the who international standard with a predefined cmv dna concentration to achieve nominal values of , and iu/ml. the reference materials were assayed in triplicate in a single run. cmv dna levels measured in bal fluid specimens closely matched those expected (the mean standard deviation for both pcr assays was < . log iu/ml for each cmv who standard concentration tested). gram stain, fungal stain and acid-fast bacilli stain were routinely performed. cytology examination (bal fluid cytospins) was performed systematically in patients attended at hcu, and nonroutinely at hlf. in all, bal fluid cytospin data were available from patients. in addition, bal fluid specimens were examined for the presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, which allows detection of rvs, or the clart® pneumovir assay (genomica, coslada, spain)-at both centers-that makes it possible to detect simultaneously rvs, as previously reported. quantitative cultures of bal specimens for bacterial isolation were performed on conventional media as recommended ; bacterial loads > cfu/ml were deemed to be clinically relevant. bal specimens were cultured on bcye-alpha agar, bd (becston dickinson) mgit® (mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. calcofluor white, blue toluidine or direct immunofluorescence staining procedures were used for detection of pneumocystis jiroveci. lung biopsy tissue specimens were not obtained. likewise, neither shell vial and conventional viral cultures for cmv detection or recovery nor direct fluorescent antibody testing for cmv detection were performed. [ ] . acyclovir/valacyclovir prophylaxis against herpes simplex and varicella zoster viruses was given to all patients as previously described. , - all patients received standard antibacterial and antifungal prophylaxis. , - definitions cmv dnaemia and pulmonary cmv dna shedding were defined as the detection of cmv dna (at any level) in one or more plasma or bal fluid specimens, respectively. proven cmv pneumonia categorization required histopathological evidence (i.e., viral inclusions and immunohistochemical staining) in biopsy (autopsy) lung tissue. cmv pneumonia diagnosis was reasonably excluded if cmv-induced cytopathogenetic effect was not observed in post-mortem (autopsy) lung tissue (data were available from patients) or bal fluid cytospins (data available from patients), there was a lack of chest x ray and computed tomography (ct) evidence consistent with cmv pneumonia (ie. reticulonodular infiltrates, the presence of bilateral ground-glass opacities, air-space opacities, small centrilobular nodules < cm, and absence of larger nodules -see , for review -) and alternative diagnoses that could account for the signs and symptoms, particularly when the presence of other significant bacterial, virus or fungal pathogens was demonstrated. the clinical and radiographic response to targeted antimicrobial therapy (for bacterial, fungal and non-cmv viral infections) was also considered to be against the involvement of cmv. the probable cmv pneumonia category, according to recent criteria was not considered herein, since culture-based bal fluid testing was not performed and no diagnostic cmv dna cut-off level has been definitely established. diagnosis of proven, probable, and possible invasive fungal infection and pneumocystis jiroveci pneumonia was achieved following consensus criteria. , acute graft versus host disease (agvhd) was diagnosed and graded according to standard criteria. frequencies were compared using the χ test (fisher exact test) for categorical variables. differences between medians were compared using the mann-whitney u test (for two independent variables) or the kruskal-wallis test (for more than two independent variables). cumulative incidence plots of mortality from pneumonia were generated with the graphpad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test. cox proportional hazards models were used to evaluate unadjusted and adjusted hazard ratios (ahrs) for mortality attributable to pneumonia, as previously reported. for multivariate analyses, only variables with parameter estimates showing a p value ≤ . in the univariate analyses were included. two-sided exact p values are reported and p values ≤ . were considered statistically significant. the data were analyzed with the spss (version . ) statistical package. table shows relevant demographic and clinical characteristics of the patients in the cohort. bal fluid samples were obtained at a median of . days after allo-hsct (range days to five years). specific details on the microbiological yield of bal fluid specimens are shown in table . proven cmv pneumonia was diagnosed in patients ( . %) on the basis of histopathological and immunohistochemistry findings in lung autopsy tissue. in the remaining episodes, pneumonia was attributable to bacteria in cases ( . %), and to one or more viruses in ( . %). there were invasive aspergillosis infection cases ( . %), these being categorized as possible ( n = ), probable ( n = ), or proven ( n = ). mixed infections were documented in patients ( . %). the individual pathogenetic contribution of each detected or cultured microbial agent to pneumonia was not attempted to be settled. twenty-three pneumonia cases ( . %) were deemed not to have an infectious cause on the basis of clinical and radiographic data and the lack of detection of any potential pathogenic microorganism (other than cmv) in bal fluid specimens. cmv dna was detected in out of bal fluid specimens ( . %): from patients with pneumonia episodes in which potential respiratory pathogens (one or more) were either cultured or detected, and from cases deemed not to have an infectious origin. in these latter cases, the involvement of cmv as the causative agent was reasonably ruled out on clinical and radiographic grounds. cmv dna was present in bal fluid specimens from the two patients with proven cmv pneumonia; no other microorganisms were concurrently detected/cultured in these two specimens. the frequency of cmv dna detection was comparable ( p = . ), regardless of the established (or presumed) etiology of pneumonia ( table ). in episodes, cmv dna bal detection occurred in the face of an ongoing episode of cmv dnaemia (including two episodes that occurred in the setting of autopsy-proven cmv pneumonitis), and isolately in the remaining episodes. clinical and laboratory characteristics of patients ( n = ) in whom cmv dna was co-detected in bal and plasma specimens in the absence (presumed) of cmv pneumonitis ( n = episodes) merit special attention, as cmv lung disease usually occurs concomitantly with cmv dnaemia. , the data are shown in supplementary table . cmv pneumonitis was deemed to be unlikely in these patients owing to one or more of the following: (i) lack of typical findings in cts (in all episodes); (ii) negative bal cytospin results (in episodes); (iii) negative lung histopathology at autopsy (in table pneumonia attributable etiology and microbiological findings in bronchoalveolar lavage fluid specimens. no. (%) c according to [ ] . d according to [ ] . e including idiopathic pneumonia syndrome, bronchiolitis obliterans and cryptogenic organizing pneumonia among other causes. out of patients who died); (iv) survival of patients who did not undergo specific anti-cmv treatment courses with appropriate doses for cmv pneumonitis ( episodes); of note, no patient in this series was treated with anti-cmv drugs for cmv pneumonitis as recommended ; (v) documentation of the presence of bacteria, viruses (other than cmv) or fungal pathogens known to cause pneumonia (in episodes); (vi) clinical response (survival) to targeted anti-bacterial, anti-viral (influenza virus) or anti-fungal therapy (in episodes). nevertheless, in particular, there were four episodes in which the causative involvement of cmv raised doubts (second episode in patient , and episodes in patients , and -supplementary table . despite the fact that ct scans were not suggestive of cmv pneumonitis, no alternative microbial etiology was documented and all these patients died (pneumonia was the attributable cause of death). in addition, cmv dna was detected at high levels in both bal (ranging from to , iu/ml) and plasma (ranging from to , iu/ml) specimens. one of these patients (patient ) had negative bal cytospin results. in turn, cmv dnaemia was documented in cases in the absence of cmv dna detection in bal fluid samples. recipient cmv seropositivity and treatment with corticosteroids were associated with detection of cmv dna in bal fluid specimens in univariate analyses (supplementary table ). overall, the median cmv dna load in bal fluid specimens from patients categorized as not having cmv pneumonia was iu/ml (range, - , iu/ml). this magnitude was greater ( p = . ) in specimens analyzed at hlf (median, iu/ml; range, - , iu/ml) than in those being tested at hcu (median, iu/ml; range, - , iu/ml) (supplementary fig. ) . the cmv dna load was > iu/ml in pneumonia episodes and < iu/ml in the remaining ( table ). the cmv dna load in bal fluid in the two proven cmv pneumonia cases was and , iu/ml. a trend towards higher cmv dna loads in bal fluid specimens was observed ( p = . ) in episodes in which cmv dnaemia was detected concurrently ( iu/ml; range, - , iu/ml vs. iu/ml; range, - , iu/ml in its absence). cmv dna loads in bal fluid specimens were comparable ( p = . ), irrespective of the etiology (attributable) of pneumonia ( fig. , and supplementary table ) . overall, there was a trend towards an inverse correlation between the level of immunosuppression, as inferred by the immunodeficiency index (isi) -see footnotes in table -, and the cmv dna load quantified in bal specimens (rho, − . ; p = . ). overall, patients ( %) were under anti-cmv therapy at the time of bal sampling (prophylaxis, n = ; preemptive therapy, n = ). among those with cmv dna detectable in bal fluids, ( %) were receiving anti-cmv therapy (preemptive therapy, n = , and antiviral prophylaxis, n = ). the cmv dna load in bal was significantly higher in patients who were under antiviral therapy (median, iu/ml; range, - , iu/ml vs. median, iu/ml; range, - iu/ml; p = . in non-treated patients). two out of the patients with cmv dna detected in bal fluids and no concurrent cmv dnaemia were treated with i.v. ganciclovir. forty-six patients ( %) died within days after bal sampling, including one patient with proven cmv pneumonia. the cause of death was deemed to be related to the pneumonia episode in patients ( %). the cumulative incidence of pneumoniaattributable mortality was comparable across the different etiological categories ( fig. ) . we investigated whether the presence of cmv dna in bal fluid specimens was associated with increased mortality, and found this not to be the case. nevertheless, roc curve analysis enabled to determine a cut-off cmv dna level in bal fluid identifying those patients with increased mortality; this threshold was iu/ml (sensitivity: . %; % ci . - . %; specificity, . %; % ci, . − . %) (supplementary fig. ). adjusted cox models including in addition to this variable a number of others that could have had an impact on pneumoniaattributable mortality identified the detection of a cmv dna load in bal fluid specimens at levels > iu/ml, treatment with corticosteroids (at any dose) and lymphocyte counts < . × /l, the [ ] . b the cut-off was established by roc analysis (not shown). cumulative incidence of mortality attributable to pneumonia complications by day after bronchoalveolar lavage fluid testing according to the etiology. cumulative incidence plots were generated with the graphpad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test (the p value is shown). latter two at the time of bal sampling, as the parameters that were independently associated with pneumonia-attributable mortality ( table ) . moreover, as shown in fig. , these factors appeared to exert a synergistic impact on mortality. the definitive abandonment of traditional culture-based procedures for cmv testing in most laboratories and the non-availability of lung biopsy specimens for histopathological and immunohistochemistry examination make the diagnosis of proven or probable cmv pneumonia elusive nowadays. moreover, no cut-off value for cmv dna load in bal fluid specimens discriminating between cmv pneumonia and pulmonary cmv dna shedding in allo-hsct recipients has been established at the present time. the assessment of the performance of quantitative cmv dna pcr testing for the diagnosis of cmv pneumonia faces several difficulties including: (i) the low incidence of this clinical event, (ii) the lack of a normalized procedure for cmv dna pcr testing on bal fluids, (iii) the non-negligible possibility of miscategorization of pneumonia cases as either being causally linked or unrelated to cmv when using bal fluid specimens, or even lung tissue material, for cmv diagnosis, (iv) the persistence of a large variability of cmv dna loads provided by different real-time pcr assays, - despite their calibration to the who international standard for cmv dna and, as already mentioned, (v) the relegation of virological procedures for cmv detection in clinical specimens. inevitably, all these drawbacks were encountered in this study, so that we aimed not to establish a diagnostic cut-off, but rather to assess the range of cmv dna loads quantifiable in bal fluid specimens from patients in whom the causal involvement of cmv was highly unlikely or could be reasonably discarded; this, having in mind that we surely incurred cmv pneumonia infradiagnosis. nevertheless, we perceived this limitation as not being an insoslayable obstacle to our purpose fig. . cumulative incidence of mortality attributable to pneumonia complications by day after bronchoalveolar lavage fluid testing according to the presence of one or more factors associated with mortality in multivariate cox models (cmv dna load in bal fluid iu/ml, treatment with corticosteroids and total lymphocyte counts < . × /l). cumulative incidence plots were generated with the graph-pad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test (the p values for comparisons are shown. as: (i) cmv is unlikely to be the etiological agent of more than % of pneumonia cases among allo-hsct recipients who undergo routine bronchoscopy, and (ii) survival of patients with cmv pneumonia who do not undergo specific anti-cmv treatment with appropriate doses of (val)ganciclovir or foscarnet is highly unlikely, provided the high rate of cmv pneumonia-associated mortality. we retrospectively reviewed clinical and microbiological data from patients who underwent routine quantitative cmv pcr testing on bal fluid specimens at two transplant centers in our city. we deliberately chose not to include archived bal specimens in our series because of the lack of data on the impact of cryopreservation on cmv dna load quantitation in this sample type. several findings arose from the present study. first, we confirmed previous observations - indicating that detection of cmv dna in bal fluid specimens using highly-sensitive pcr assays is a very common finding in allo-hsct patients with pneumonia, irrespective of the definitive etiological diagnosis. in our series, cmv dna was detected in more than one third of bal fluid samples, the frequency of detection varying between % in patients seemingly with not having an infectious pneumonia and % in patients with mixed infections. in our series, recipient cmv seropositivity and treatment with corticosteroids were associated with detection of cmv in bal fluid specimens. of interest, the two patients with proven cmv pneumonia had cmv dna detectable in bal fluid. second, we found a wide range of cmv dna loads measured in bal fluid specimens from patients with pneumonia in whom cmv causality was unlikely or reasonably ruled out attending to clinical (including therapeutic response to nonanti-cmv drugs), radiographic, lung autopsy histopathology or bal fluid cytology (in some patients) and microbiological criteria. interestingly, median cmv dna loads were comparable irrespective of the nature and the number of co-detected microorganisms (at both participating centers), and were overall higher in the presence of concurrent dnaemia. as for the latter observation, in agreement with boeckh et al., we found this not to be due to pulmonary hemorrhage (not shown). overall, cmv dna loads in bal fluid specimens processed at hlf were of greater magnitude than those analyzed at hcu. since cmv dna loads produced by the argene pcr assay are slightly lower than those measured by the abbott pcr assay (not shown), differences in the net state of immunosuppression of patients at the time of bal sampling across centers, as reflected by the immunodeficiency score index (higher for hlf patients), may account for this observation. in fact, a trend towards an inverse correlation was found between the isi score and the cmv dna load quantified in bal specimens. in a recent study, a cmv dna load cut-off of iu/ml in bal fluid was found to have a positive predictive value of ∼ % for the presence of probable cmv pneumonia (considering a prevalence of this event of % among patients at risk and undergoing bal testing). tan et al., in contrast, found cmv dna levels in bal fluid samples to have a limited value to distinguish between cmv and non-cmv pneumonia cases. it is pertinent to mention here that the above threshold is between one and two log lower than those tentatively proposed for diagnosing cmv pneumonia in lung transplant recipients. - interestingly, control patients with non-cmv pneumonia in the boeckh study showed a median cmv dna load of log iu/ml (iqr, - . iu/ml), with cmv dna levels between and iu/ml in roughly % of cases and > iu/ml in % of them. here, the opposite was true, with nearly % of bal fluid samples from patients with non-proven cmv pneumonia having cmv viral loads > iu/ml, of which % had > iu/ml. it is worth highlighting that these figures were comparable at both participating centers, despite the above-referred differences in cmv dna loads provided by pcr assays used at each center. to gauge the potential relevance of these data, it must be taken into consideration that in nearly % of pneumonia episodes in our cohort, bal sampling was performed while patients were under anti-cmv therapy ( > days), whereas in the aforementioned study, only % of patients with cmv pneumonia and % of patients with non-cmv pneumonia had been treated with antivirals for at least two days. despite this fact, higher cmv dna loads in bal fluid specimens were quantified in the current study, likely reflecting major differences regarding the dnaemia cut-off triggering the inception of antiviral therapy between these studies (much higher in the current cohort). in fact, in this series, the median cmv dna load in bal was significantly higher in patients who were under antiviral therapy than in non-treated patients. as stated above, the limited number of proven cmv pneumonia cases in our series precluded any attempt to establish a diagnostic cmv dna load cutoff; nevertheless, in light of the data presented herein, a threshold value of iu/ml is unlikely to be discriminative between cmv pneumonia and pulmonary cmv dna shedding in our setting. in this sense, we fully support the idea that the magnitude of such a diagnostic cut-off is likely to vary depending upon the patient's characteristics, the bal procedure and processing, the assay used for cmv dna quantitation and the severity of cmv pneumonia at the time of sampling. our data should be interpreted with caution as the exclusion of cmv as the probable causative agent can be judged as dubious in some cases provided that no virological methods were used to investigate the presence of cmv in bal fluid specimens. in particular, there were episodes occurring in patients who died, in whom cmv dna was detected at high levels in both bal and plasma specimens ( > iu/ml) and no alternative microbial etiology was documented. nevertheless, the conclusions drawn on the basis of the overall dataset stood when cases in which little or no doubt existed on the lack of involvement of cmv (i.e. bacterial pneumonia, tuberculosis.) were analyzed separately (representative examples are shown in supplementary table ) . cmv is a highly pro-inflammatory and immunosuppressive virus; as such it may act as a synergistic co-pathogen in the absence of documented cmv-induced cytopathogenicity, and may have a relevant impact on patient outcome. in this sense, we found that the presence of cmv dna in bal fluid specimens at levels > iu/ml, in addition to receipt of corticosteroids and low lymphocyte counts at the time of bal sampling, was associated with increased pneumonia-attributable mortality in cox multivariate models. the relative scarce number of pneumonia cases in which bal specimens had cmv dna loads > iu/ml did not allow to investigate whether this apparent effect exhibited a dose-response pattern. again, this finding must be interpreted cautiously, as in order to avoid overfitting, cox models were not adjusted to a number of factors that may have had an impact on mortality (i.e., adequacy of antimicrobial treatment, severity of pneumonia, among others). the limited size of the current cohort also precluded any meaningful statistical analysis evaluating the impact of cmv dna load in bal at each center, separately. further studies are urgently needed to validate this observation, since this subset of patients may benefit from short courses of anti-cmv therapy. limitations of the current study, in addition to the ones highlighted above, are its retrospective nature, the potential biased selection of patients requiring bronchoscopy for the etiological diagnosis of pneumonia, the lack of normalization of cmv dna loads in bal fluids to cellular dna content (although this was reported to be expendable in a previous study ) and the use of different dna extraction platforms and real-time pcr assays for cmv dna quantitation. nevertheless, this study has several strengths, including the inclusion of consecutive specimens, the use of fresh bal fluid specimens and highly-sensitive real-time pcr assays for cmv dna load quantitation, and the performance of a comprehensive and systematic evaluation of specimens for the presence of rvs pathogens using molecular assays. in summary, our study highlights the difficulty in establishing universal cmv dna load thresholds in bal fluid specimens for distinguishing between cmv pneumonia and pulmonary cmv dna shedding. despite this, the potential impact of the presence of cmv in bal fluid specimens on pneumonia-attributable mortality observed herein merits to be further investigated. to this end, only large multicenter prospective studies using consensus protocols for cmv dna pcr bal testing and conventional 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institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients clinical manifestations of graft-versus-hostmdisease in human recipients of marrow from hl-a-matched sibling donors are we there yet? impact of the first international standard for cytomegalovirus dna on the harmonization of results reported on plasma samples spanish society for infectious diseases and clinical microbiology quality control study group would kinetic analyses of plasma cytomegalovirus dna load help to reach consensus criteria for triggering the initiation of preemptive antiviral therapy in transplant recipients progress in quantitative viral load testing: variability and impact of the who quantitative international standards human cytomegalovirus load in plasma and bronchoalveolar lavage fluid: a longitudinal study of lung transplant recipients clinical utility of cytomegalovirus viral load in bronchoalveolar lavage in lung transplant recipients relationship between cytomegalovirus dna load in epithelial lining fluid and plasma of lung transplant recipients and analysis of coinfection with epstein-barr virus and human herpesvirus in the lung compartment preemptive therapy for systemic and pulmonary human cytomegalovirus infection in lung transplant recipients cytomegalovirus viral load in bronchoalveolar lavage to diagnose lung transplant associated cmv pneumonia immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections this research was supported by a grant ( / ) from fis ( fondo de investigaciones sanitarias, ministerio de sanidad y consumo, spain).estela giménez holds a río hortega research contract from the carlos iii health institute (ref. cm / ). none. supplementary material associated with this article can be found, in the online version, at doi: . /j.jinf. . . . key: cord- -d re r authors: boonyaratanakornkit, jim; vivek, meghana; xie, hu; pergam, steven a; cheng, guang-shing; mielcarek, marco; hill, joshua a; jerome, keith r; limaye, ajit p; leisenring, wendy; boeckh, michael j; waghmare, alpana title: predictive value of respiratory viral detection in the upper respiratory tract for infection of the lower respiratory tract with hematopoietic stem cell transplantation date: - - journal: j infect dis doi: . /infdis/jiz sha: doc_id: cord_uid: d re r background: hematopoietic cell transplant (hct) recipients are frequently infected with respiratory viruses (rvs) in the upper respiratory tract (urt), but the concordance between urt and lower respiratory tract (lrt) rv detection is not well characterized. methods: hematopoietic cell transplant candidates and recipients with respiratory symptoms and lrt and urt rv testing via multiplex pcr from to were included. logistic regression models were used to analyze risk factors for lrt rv detection. results: two-hundred thirty-five hct candidates or recipients had urt and lrt rv testing within days. among subjects ( %) positive for a rv, % ( of ) had discordant sample pairs. forty percent ( of ) of discordant pairs were positive in the lrt but negative in the urt. discordance was common for adenovirus ( %), metapneumovirus ( %), rhinovirus ( %), and parainfluenza virus type ( %); respiratory syncytial virus was highly concordant ( %). likelihood of lrt detection was increased with urt detection (oods ratio [or] = . ; % confidence interval [ci], . – ) and in cytomegalovirus-positive recipients (or = . ; % ci, . – . ). conclusions: high rates of discordance were observed for certain rvs. bronchoalveolar lavage sampling may provide useful diagnostic information to guide management in symptomatic hct candidates and recipients. respiratory virus infections are a major cause of mortality and morbidity in hematopoietic cell transplant (hct) recipients [ ] [ ] [ ] . although symptomatic patients are frequently tested for viruses in the upper respiratory tract (urt), lower respiratory tract (lrt) testing with bronchoscopy and/or bronchoalveolar lavage (bal) is done less frequently, often only when prompted by clinical deterioration or for further evaluation of findings on radiologic imaging. our study assessed the correlation between concurrent urt and lrt testing for respiratory viruses in hct pretransplant candidates and posttransplant recipients. few studies have examined differences in respiratory viral detection by polymerase chain reaction (pcr) between upper and lower tract samples. in immunocompetent children with chronic respiratory symptoms, paired nasopharyngeal (np) aspirate and bal samples showed discordance in approximately one third of patients, with positive np aspirate/negative bal discordance being most common [ ] . in a small study of adults, the majority of which were hct recipients or had a hematologic malignancy, with matched np and bal specimens, pcr-based np testing for respiratory viruses in patients with clinical evidence of lrt disease had a high negative predictive value (npv) and a lower positive predictive value (ppv) [ ] . a larger study that included mostly immunocompromised patients concluded that if a pathogen (a respiratory virus or of bacterial pathogens detected by a multiplex pcr panel) was already identified from an np sample, bal testing is unlikely to provide additional information; however, a significant number ( %) of subjects had a pathogen detected in the bal without a positive np sample [ ] . likewise, in a study of lung transplant recipients, viral detection exclusively in the lrt was reported, and thus the authors caution on the use of urt sampling alone to rule out lrt infection in this population [ ] . our study aimed ( ) to describe discordance in hct candidates and recipients and ( ) to characterize specific viral, patient, and treatment risk factors that are associated with lrt detection. in addition, using quantitative pcr methodology, we aimed to define the role of viral load in respiratory virus lrt detection. we retrospectively identified hct pretransplant candidates (within days of hct) and posttransplant recipients who underwent a bal ± and ± days from an np aspirate with testing of respiratory specimens by multiplex pcr testing for [ ] . in total, exactly hct recipients underwent bal respiratory virus testing during the study period. clinically indicated bronchoscopy for lrt symptoms and/or radiographic abnormalities was determined by a pulmonologist. a bal was generally performed to either rule-in respiratory viral involvement of the lrt and/or to rule out alternative causes for a lrt process. the bals were collected per institutional standard practice using up to three -ml aliquots of normal saline. from this cohort, we then identified subjects who had urt viral pcr testing (from nasal wash or np swab) within days or within day of the bal. only the first bal per subject was included in the analysis. cycle thresholds (ct) were used as a proxy for viral load and were compared between upper and lower respiratory sample pairs that were positive for the same virus for a given subject. patient charts were reviewed for steroid use, radiology results, presence of copathogens, and potential alternate diagnoses. steroid dose was defined as the highest dose of steroid in milligrams/kilogram per day expressed in equivalent doses of prednisone in the weeks preceding the bal. computed tomography of the chest obtained within week of the bal were reviewed. we chose to categorize imaging results for the presence versus absence of a solitary nodule because patients with a solitary nodule may carry an alternative diagnosis and be at lower risk for viral lrt involvement. copathogens were defined as follows: ( ) bacterial -> colony-forming units of a gram-positive organism or any gram-negative organism on bal; ( ) viral -any other respiratory virus detected in bal by multiplex pcr or cytomegalovirus (cmv) shell vial positivity; and ( ) fungal -positive serum or bal galactomannan, bal fungal pcr positivity, or bal fungal culture positivity (excluding candida species) [ , ] . alternate diagnoses included diffuse alveolar hemorrhage based on finding progressive bloody fluid return on bal [ ] . discordance was defined as either ( ) a urt sample positive for a respiratory virus with the paired lrt sample negative for the same virus (termed positive/negative [p/n]) or ( ) a urt sample negative for a respiratory virus with the paired lrt sample positive for the same virus (termed negative/positive [n/p]). positive concordance was defined as both urt and lrt paired samples being positive for the same virus (p/p), and negative concordance was defined as both urt and lrt paired samples being negative for the same virus (n/n). the sensitivity and specificity for lrt detection by ct value in the urt was plotted by generating a receiver operating characteristic (roc) curve. the ct value for subjects with negative testing in the urt was set at , above the upper limit of assay detection, to allow inclusion of these subjects in the roc analysis. positive and negative predictive values for lrt infection were plotted as a function of all possible ct value cutpoints. logistic regression models were used to evaluate odds ratios (or) for the association between candidate risk factors and viral lrt detection. patients with more than virus detected in the urt (n = ) were excluded from the logistic regression analysis. all patients in the cohort with lrt detection of adenovirus also had adenovirus testing of the plasma by pcr for viremia. patients with disseminated adenovirus as evidenced by a positive plasma pcr at the time of lrt detection (n = ) were excluded from the logistic regression and roc analysis. variables with p ≤ . in univariable analysis were candidates for multivariable models and were retained in the models if they remained significant themselves or modified the effect of another factor (confounder). covariates evaluated as candidate risk factors for inclusion in multivariable models are listed in table . statistical significance was defined as -sided p < . . sas version . ts m (sas institute inc., cary, nc) was used for all statistical analyses. we identified subjects with a bal performed during the study period who had urt rv testing within days of the bal. table shows the demographic characteristics of the cohort. the majority of patients ( %) were between and years of age, and % were male. the majority of patients ( %) underwent allogeneic transplant. only % had a bal before hct. the median number of days between bal and nasal swab was day. table also shows the demographics for a subset of subjects in this cohort with urt and lrt testing within day of each other; the subset is largely representative of the whole cohort. among the sample pairs in the overall cohort, % ( of ) were positive for a respiratory virus in either the urt or lrt. of these, % ( of ) were concordant positive for the same virus in both upper and lower tracts (p/p). discordance was noted in % ( of ) of sample pairs. among the discordant pairs, % ( of ) were positive in the urt but negative in the lrt (p/n), and % ( of ) were negative in the urt but positive in the lrt (n/p). figure shows the distribution of concordance/discordance for pairs with at least test positive for individual viruses. in patients who underwent bal within days of an np aspirate, discordance between urt and lrt results was observed at the highest rate for hmpv ( positive pairs, % n/p and % p/n), hrv ( positive pairs, % n/p and % p/n), piv ( positive pairs, % n/p), and piv ( positive pairs, % n/p and % p/n) ( figure a ). all pairs with a positive adenovirus result were discordant ( % n/p and % p/n). respiratory syncytial virus had the highest frequency and percentage of concordant results ( positive pairs, % concordant with % p/n and no n/p pairs). similar patterns of discordance were observed when analyzing patients who underwent bal within day of an np aspirate ( figure b ) and when analyzing the subset of patients who underwent bal after a positive np aspirate (supplemental figure ) . the ct values of all viruses grouped together between the urt and lrt in concordant pairs were not significantly different when the bal was performed either ± days (Δct urt-lrt = − . , p = . ) or ± day (Δct urt-lrt = − . , p = . ) from collection of the urt specimen. the distribution of copathogens and alternate diagnoses was examined for concordant positive pairs and discordant pairs ( figure ). aspergillus fumigatus, another respiratory virus, or bacteria were the most commonly identified copathogens. no significant differences were observed in the proportion of copathogens or alternate diagnoses in subjects with discordant testing versus concordant positive results by fisher's exact test. in a univariable analysis of risk factors for lrt detection, detection of virus in the urt (or = . ; % confidence interval [ci], . - ) and recipient cmv seropositivity (or = . ; % ci, . - . ) were associated with an increased risk for lrt detection (table ). of note, factors not found to be associated with lrt detection included conditioning regimen, transplant type (allogeneic versus autologous), steroid use, presence of a solitary nodule on imaging, or lymphocyte, neutrophil, monocyte, or overall white blood cell counts. in a multivariable analysis, detection of virus in the urt (or = . ; % ci, . - ) and recipient cmv seropositivity (or = . ; % ci, . - . ) remained strongly associated with an increased risk for lrt detection ( table ). an analysis of the subset of patients who underwent bal within day of the np aspirate and a separate analysis of the subset of only hct recipients who underwent bal after transplantation yielded similar results ( supplementary tables and ) . the proportion of patients with a positive lrt sample was similar between patients with urt testing before lrt testing ( % or of ) versus after lrt testing ( % or of ). thirty-day mortality in patients with positive respiratory viral testing in the lrt was . % ( of ) compared with . % ( of ) in those with negative testing, although this difference was not statistically significant (p = . ). a roc curve was generated to summarize sensitivity and specificity of varying ct cutpoints in the urt for the presence of lrt infection. a ct cut point of . in the urt had a sensitivity of % and a specificity of %, whereas a cutpoint of . had a sensitivity of % and a specificity of % ( figure a ). using a ct cutoff of . in the urt with a prevalence of lrt infection of % ( of ) in the cohort, the ppv was % and the npv was % for lrt infection ( figure b ). with higher ct values in the urt, the ppv declined whereas the npv for lrt infection increased. for comparison, any positive test in the urt had a ppv of % and a npv of %. in the present study, we characterized the rates of discordance in respiratory viral detection between matched urt and lrt samples in a large cohort of hct candidates/recipients who underwent bal for suspected lrti and had concomitant urt testing. we demonstrate high discordance rates for hrv, hmpv, piv , and adenovirus between the urt and lrt. furthermore, we identified risk factors for detection of respiratory viruses in the lungs of hct candidates and recipients, including viral detection in the np and recipient cmv seropositivity. before the advent of molecular testing, a high rate of discordance was observed with rapid antigen detection assays which had a sensitivity of % in urt specimens and % in lrt specimens from immunocompromised adults [ ] . discordance between molecular diagnostic testing of urt and lrt specimens has also been reported in other studies, particularly in immunocompromised populations, where %- % of patients with a positive lrt specimen had a concordant urt specimen [ , , ] . the positive and npvs of urt testing were %- % and %- %, respectively. in a different study of hct recipients with hmpv or rsv detected in the lrt, % had a discordant negative test for hmpv, whereas no patients had a discordant negative test for rsv [ ] . in the present study, we found high levels of discordance with % of sample pairs among subjects with positive testing showing discordance between urt and lrt testing for any virus. although the sample sizes were too small for each virus to perform statistical testing, discordance rates were highest for hmpv, hrv, adenovirus, and piv . of note, this discrepancy was present also among patients with urt specimens obtained within day of the bal. human metapneumovirus lrt disease is associated with high mortality in hct recipients, and our results suggest that a negative urt specimen may not be sufficient to rule out lrt infection [ ] . we noted that adenovirus testing showed discordance in every subject with the virus. reactivation in other tissues followed by viremia and dissemination to the lungs may have contributed to this finding. in contrast, rsv testing showed an approximately % concordance, suggesting that patients with rsv detected in the urt and clinical/radiographic evidence of lrt involvement may be presumed to have rsv in the lrt. these patients could be treated accordingly for rsv pneumonia and also be enrolled for clinical trials. we have previously categorized lrt infection with respiratory viruses into groups depending on viral detection in the lrt, where proven lrti is defined as a positive lrt sample (bal, lung biopsy, or autopsy specimen) with radiographic abnormality, probable lrti is defined as a positive lrt sample without radiographic abnormality, and possible lrti is defined as a positive urt sample with radiographic abnormality but no lrt sampling. we have shown that subjects with possible lrti have outcomes more similar to urt infection for both piv and rsv, and that subjects with proven/probable lrti have worse outcomes including need for oxygen, oxygen-free days, and mortality [ , ] . these results suggest that lrt testing to stratify patients into possible versus proven/probable lrti can provide useful prognostic information in hct recipients. this may become increasingly important because new antivirals are in development, many of which are being evaluated depending on the site of infection (upper versus lower tract). our study included patients positive for a respiratory virus in the lrt but negative in the urt. five viruses, namely, adenovirus influenza a, piv , piv , hmpv, and hrv, had n/p discordance. thus, proximal urt testing did not identify the lrt pathogen in these cases, including viruses that may warrant treatment with current antivirals (influenza a and adenovirus) as well as viruses for which specific antiviral therapy is being developed (piv and hmpv). the probability of detecting virus in the lrt was increased in patients with virus detected in the urt. other studies have found an association between higher respiratory viral loads and more severe disease manifestations [ ] [ ] [ ] . we found that lower ct values in the urt were associated with higher ppvs for lrt infection. however, the npv appeared to plateau such that the npv at a ct value of . was similar to the npv of a negative test in the urt ( % vs %, respectively). it is important to note that because even a negative pcr in the urt is not fully predictive, ct values for viruses detected in the urt cannot be used on their own to rule out lrt involvement. we also found that cmv-seropositive hct recipients had an increased risk for lrt respiratory virus detection. we have previously reported recipient cmv seropositivity as a risk factor for respiratory virus acquisition and progression to lrt infection after hct [ , ] . another study reported an association between cmv reactivation and rsv infection after hct with the development of severe pneumonia [ ] . cytomegalovirus seropositivity and reactivation have been associated with increased morbidity, mortality, and graft-versus-host disease after hct [ ] [ ] [ ] . the pathogenesis of cmv infection and disease is complex with several immunomodulating interactions between cmv and the immune system, including effects on human leukocyte antigen expression and cytokine production (reviewed in reference [ ] ). our findings here suggest that increased risk of lrt infection may be another indirect effect of cmv. furthermore, we observed a trend towards decreased risk of detecting virus in the lrt in patients transplanted between and and a trend towards decreased risk in patients transplanted between and compared to and . it is possible this could have been secondary to a decrease in virus detected in the urt in the later years ( % between and , % between and , and % between and ) because a negative result in the urt was strongly associated with a lower risk for lrt detection. this reduction in urt respiratory viral detection may also have been secondary to improvements in infection control practices. alternatively, the trend towards reduced risk of lrt detection in later years may be a reflection of practice changes either with ( ) delaying transplants in patients with positive testing for respiratory viruses in the urt or ( ) fewer bronchoscopies being performed now compared to in the past [ ] . this could have led to a sampling bias in more recent years in which a bronchoscopy was more often performed when lung disease due to an alternative, nonrespiratory viral, process was suspected. we also sought to understand the differences in viral load observed in concordant upper and lower samples. there was no significant difference between viral loads in the upper versus lower tract when analyzing all viruses together in patients with a bal within days or day of urt testing. one argument against early bronchoscopy to test for viral lrt involvement includes the notion that viruses in the urt may be "pushed" into the lrt during the procedure itself. lower respiratory tract positivity could also simply reflect upper tract secretions that are aspirated during the procedure, similar to the finding of oral flora in bacterial cultures of a bal. the finding of several cases of positive testing in the nose yet negative testing in the lungs argues against the "pushing down" of viruses from the urt to the lrt during bronchoscopy and against the detection of viruses in the lrt as an artifact of contamination from the urt during bronchoscopy. the sample size was too small to support analysis of ct values for individual viruses. our study has several limitations. first, even though our sample size was relatively larger than other studies of immunocompromised patients with paired urt and lrt sampling, we could not evaluate risk factors for discordance for individual viruses. second, data were collected retrospectively, and, therefore, need for sampling of the urt and lrt was determined by the clinician. the patient population was limited to those who underwent bal within or days of an np aspirate because viral detection in the lrt was an outcome measure, and therefore risk factors may differ with patients who undergo only urt testing. third, our primary analysis focused on hct recipients who had undergone bal within days from an np aspirate. this could be considered too long a time period. more important, however, the results from this analysis were similar to that for patients who had undergone bal within day of an np aspirate. fourth, differences in sampling from the lrt by bal and urt by swabbing may contribute to differences in ct values. the higher collection volume for bals compared with np aspirates (approximately vs ml) would generally lead to higher ct values in lrt specimens due to greater dilution. fifth, antiviral therapy in patients with influenza or rsv detected in np aspirates may have affected the detection of these viruses in the bal. however, we found that almost every case of rsv infection was concordant and only case of influenza was discordant p/n. sixth, because copathogens and alternative diagnoses were identified in many patients, we cannot definitively conclude whether lrt symptoms, signs, or radiographic abnormalities were caused specifically by the respiratory virus, the copathogen, and/or a concomitant noninfectious process. finally, ct values may not be as generalizable between different assays compared with a true viral load measured in copies/milliliter. even though most currently available commercial pcr assays do not include ct values, our study shows the predictive value of these results for lrt infection. in summary, our data demonstrate discordance between urt and lrt respiratory virus detection for several common respiratory viruses. we suggest that early lrt viral testing could provide useful diagnostic information that may affect management of respiratory viral infections in certain hct candidates and recipients. the challenge of respiratory virus infections in hematopoietic cell transplant recipients mortality rates of human metapneumovirus and respiratory syncytial virus lower respiratory tract infections in hematopoietic cell transplantation recipients significant transplantation-related mortality from respiratory virus infections within the first one hundred days in children after hematopoietic stem cell transplantation respiratory virus detection in nasopharyngeal aspirate versus bronchoalveolar lavage is dependent on virus type in children with chronic respiratory symptoms predictive value of testing nasopharyngeal samples for respiratory viruses in the setting of lower respiratory tract disease filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients comparison of conventional and molecular 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supplemental oxygenfree days in hematopoietic cell transplant recipients with respiratory syncytial virus viral load drives disease in humans experimentally infected with respiratory syncytial virus correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients respiratory virus pneumonia after hematopoietic cell transplantation (hct): associations between viral load in bronchoalveolar lavage samples, viral rna detection in serum samples, and clinical outcomes of hct respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection human rhinovirus infections in hematopoietic cell transplant recipients: risk score for progression to lower respiratory tract infection virus infection facilitates the development of severe pneumonia in transplant patients with hematologic malignancies pre-transplant cytomegalovirus (cmv) serostatus remains the most important determinant of cmv reactivation after allogeneic hematopoietic stem cell transplantation in the era of surveillance and preemptive therapy how we treat cytomegalovirus in hematopoietic cell transplant recipients increased transplant-related morbidity and mortality in cmvseropositive patients despite highly effective prevention of cmv disease after allogeneic t-cell-depleted stem cell transplantation cytomegalovirus: shape-shifting the immune system clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant we thank jane kuypers for help with quantitative polymerase chain reaction analysis and zach stednick and chris davis for help with data collection and management.author contributions. j. b. and m. v. designed and performed the research, collected data, analyzed data, and wrote the manuscript; h. x. and w. l. performed statistical analyses, generated tables and figures, and critically reviewed the manuscript; s. a. p. and g. 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.supplemental figure . results of upper respiratory tract (urt) and lower respiratory tract (lrt) sample testing among patients with positive urt testing before undergoing bronchoalveolar lavage (bal). concordance or discordance is shown by specific virus (represented as result from urt/ lrt with n = negative and p = positive). data are shown for subjects with a bal ± days from the urt test. adeno, adenovirus; flua, influenza a; flub, influenza b; hcov, human coronavirus; hmpv, human metapneumovirus; piv, parainfluenza viruses - ; hrv, human rhinovirus; rsv, respiratory syncytial virus. key: cord- -tmr ieg authors: gallucci, marcella; pedretti, melissa; giannetti, arianna; di palmo, emanuela; bertelli, luca; pession, andrea; ricci, giampaolo title: when the cough does not improve: a review on protracted bacterial bronchitis in children date: - - journal: front pediatr doi: . /fped. . sha: doc_id: cord_uid: tmr ieg chronic cough is defined as a daily cough that persists longer than weeks. protracted bacterial bronchitis (pbb) is a common cause of chronic wet cough in preschool children with no symptoms or signs of other specific causes, and resolution usually follows a -week course of an appropriate oral antibiotic. the diagnosis is mainly clinical; generally, no instrumental examinations are necessary. the most common bacteria found in the bronchoalveolar lavage (bal) of subjects with pbb include haemophilus influenzae, streptococcus pneumoniae, and moraxella catarrhalis. nowadays, there is no certain evidence of the role of viruses in pbb pathogenesis even though different types of viruses have been detected in bal from children with pbb. airway malacia is commonly found in children with pbb; conversely, there is no correlation with any type of immunodeficiency. amoxicillin-clavulanate acid is the most commonly used antibiotic, as first-line, prolonged therapy (longer than weeks) is sometimes required to cough resolution. when the wet cough does not improve despite prolonged antibiotic treatment, an underlying disease should be considered. moreover, there are several hypotheses of a link between pbb and bronchiectasis, as recent evidences show that recurrent pbb (> episodes/years) and the presence of h. influenzae infection in the lower airways seem to be significant risk factors to develop bronchiectasis. this underlines the importance of a close follow-up among children with pbb and the need to consider chest computerized tomography (ct) in patients with risk factors for bronchiectasis. in this brief review, we summarize the main clinical and pathogenetic findings of pbb, a disease that may be related to a relevant morbidity and decreased quality of life during the pediatric age. chronic cough in childhood is related to a considerable morbidity and a decreased quality of life (qol) scores, affecting the child's sleep, the ability to play, and the school performance ( ) . it may also cause a state of anxiety for parents. nevertheless, the real impact of chronic cough on qol is difficult to quantify ( ) . both generic health-related (pedsql) and chronic cough-specific (pc-qol) qol scores among children with pbb are similar to those of children with other respiratory disease such as asthma or bronchiectasis ( ) . etiologies of chronic cough include several and heterogenous disease such as asthma, upper airway cough syndrome, and protracted bacterial bronchitis (pbb) ( ) . chronic wet cough may also be a symptom of a chronic suppurative airway disease including bronchiectasis ( ) . pbb clinical condition was first described by marchant et al. in an australian study among children with a history of chronic wet cough lasting more than weeks, a positive culture of a respiratory pathogen on bal (bacterial growth ≥ cfu/ml in bal) obtained during a flexible bronchoscopy and a clinical response to weeks treatment with antibiotics (amoxicillinclavulanate acid) ( ) ( table ) . currently, this definition has been reclassified as pbb-micro, and new diagnostic criteria have been developed on the basis of clinical symptoms, thus eliminating the need for bal, not performed routinely among the pediatric population. according to the european respiratory society (ers) guidelines new definition, pbb-clinical is based on all three of the following criteria: "presence of chronic (> weeks' duration) wet or productive cough; absence of symptoms or signs (i.e., specific cough pointers) suggestive of other causes of wet or productive abbreviations: ba, bronchial aspirate; bal, bronchoalveolar lavage; clds, cystic lung diseases; ct, computerized tomography; ger, gastroesophageal reflux; nthi, haemophilus influenzae non-typeable; pbb, protracted bacterial bronchitis; qol, quality of life; uacs, upper airway cough syndrome. cough ( table ) ; cough resolution following a - -week course of an appropriate oral antibiotic" ( , ) . the following additional definitions are used in clinical practice: pbb-extended is pbb-micro or pbb-clinical requiring weeks antibiotic treatment for cough resolution; recurrent pbb is used to define recurrent episodes (> per year) of pbb ( table ) ( ) . according to the american college of chest physicians (chest) methodological guidelines, too, the definition of microbiologically based pbb (or pbb-micro) should be used "for children aged ≤ years with pbb with lower airway (bronchoalveolar lavage or sputum) confirmation of clinically important density of respiratory bacteria (≥ cfu/ml), " in order to differentiate it from clinically based pbb ( ). we know that pbb is a common cause of persistent wet cough in preschool children aged - years worldwide (although sometimes it may affect even older subjects). it is diagnosed in - % of children consulting a pulmonary specialist. these data are confirmed by two main studies: the first is a prospective multicenter study on the cause of chronic cough including children aged < years (mean age of . years) recruited from five australian major hospitals and three rural-remote clinics newly referred with chronic cough, where it was discovered that the main cause ( %) was pbb ( ) . the latter more recent study included children aged < (mean age of . ± . years), admitted to the pediatric department for chronic cough. among these patients, the most common final diagnoses were asthma ( . %), asthma-like symptoms ( %), pbb ( . %), and upper airway cough syndrome ( . %) ( ) . for these reason, in the clinical practice, it is important to know this lung disease in order to start an appropriate therapy before the associated complications arise. as mentioned above, the most frequent symptom in children with pbb is persistent wet cough. generally, the median age ranges from . to . years even though pbb can occur also later (> years) ( ) . frequently, there is neither a correlation with upper airway inflammation such as otitis or sinusitis nor signs of underlying chronic suppurative lung disease (csld) such as digital clubbing, chest wall deformity, and auscultatory wet sound ( , ) . the prevalence of atopic features is similar to children without pbb, and no specific correlation with the exposure to tobacco smoke has been evidenced ( , ) nevertheless, tobacco exposure is known to be a risk factor for the development of chronic respiratory diseases ( ) . although generally parents report wheezing, auscultatory feedback is rare and more frequently "rattling chest" and crackles are heard ( ) . sometimes, the symptoms of pbb are confused with those of asthma because of similar elements, and occasionally, they could coexist. what differentiates the two pictures are mainly the type of cough (wet in pbb and often dry and/or nocturnal in asthma) and the response to antibiotic treatment in pbb. if a child has chronic wet cough and suspected asthma not responding to rescue and background medications, empiric treatment for pbb should be assessed ( ) . therefore, diagnosis of pbb is mainly clinical; generally, no instrumental examinations are needed (figure ). among children undergoing chest radiograph, there are no significant changes, so the examination is normal in most cases (sometimes the chest imaging shows only peribronchial changes) ( , ) . when performed, lung functional test results are usually normal ( ) . although pbb may coexist with other diseases such as asthma, no studies assessing objective reversible airflow limitation are available to date ( ) . ct scan should only be performed following a treatment failure to evaluate the possible presence of underlying bronchiectasis ( ) . bal with a flexible bronchoscope from lower airways should be performed in cases of relapse after three courses of antibiotic; however, the timing is also to be assessed with the parents ( ) . according to ers statement, usually bal is carried out in the most affected lung area (identified radiologically and/or endoscopically) ( ) . in infants, it is often easier to perform bal in the right lower lobe being, along with lingula, the preferred site because these areas offer better fluid recovery ( , ) . bal is generally not well-tolerated, and although it is a safe procedure, it may cause hypoxemia; therefore, a recent study compared bal and bronchial aspirate (ba) to investigate if the latter would bring to similar results ( ) . both bal and ba cultures provided the same result among the majority of patients ( %). differences affecting the choice of treatment were found just in a small number of subjects with pbb ( % overtreated, % undertreated, % would have received a different therapy). the study concludes that bal still remains the gold standard, even though ba could be considered in cases where bal is not tolerated, considering that the results are overlapping in the majority of cases ( ) . in contrast to the above, bts guidelines suggest that, among children with pbb, underlying conditions should be excluded, and a sputum culture should be performed before the diagnosis ( ) . likewise, when a wet cough persists after weeks of appropriate antibiotics, chest guidelines also suggest performing "further investigations (e.g., flexible bronchoscopy with quantitative cultures and sensitivities with or without chest computed tomography)" ( ). several studies documented haemophilus influenzae as the most common bacteria found in the bal of subjects with pbb ( - %), with high bacterial loads (≥ cfu/ml) ( ). most h. influenzae are non-typeable (nthi) strains representing different genotypes ( ) . streptococcus pneumoniae ( - %) and moraxella catarrhalis ( - %) follow with variable percentages among different studies ( ) . finally, it should be noted that polymicrobial infections involving more than one pathogenic bacterium have been reported in bal of children with pbb ( - %) ( , , ) . different studies examined the lower airway microbiota of children with pbb. the first study did not reveal significant differences regarding the microbiota composition between children with bronchiectasis, cystic fibrosis, and pbb; the core microbiota was superimposable with predominance of h. influenzae and oral aerobic and anaerobic (as prevotella melaninogenica) ( ) . this contrasted with the data found among adults and suggested that a chronic airway infection starts in a similar way with inadequate airway clearance of normal microbiota, but as time passed, the microbiota in these disease groups progressively diverge from one another as a result of antibiotic drugs and (maybe) as the consequence of the underlying disease ( ) . a second study conducted in found some significant differences between the microbiota of the upper and lower airways between children with pbb, bronchiectasis, and controls ( ) . in , a team of uk researchers compared protected brushing of healthy controls and children with pbb, finding that the microbiota of the latter was less different in terms of richness and evenness. bacterial communities in children with pbb were dominated by proteobacteria, and indicator species analysis showed that haemophilus and neisseria were significantly associated with the patient group ( ) . a more recent study found out that in bal of children with pbb, one or more respiratory pathogens were detected. moreover, children with pbb showed that a higher bal bacterial biomass strongly correlated with neutrophilic inflammation. pbb-microbiota were different to control-microbiota and clustered into four distinct microbiota patterns where respiratory pathogen or other microbiota species (e.g., prevotella) have been detected ( ) . the cultures of respiratory pathogens, inflammatory markers, and bal bacterial biomass were found to not be associated with this variation in alpha diversity among subjects with pbb. this suggest that inflammation and increased bacterial biomass in pbb cannot be caused only by single pathogenic species ( ) . differences between the data from these two recent studies may be due to different average age of the patient cohort, geographical differences, or contamination of the bal with upper respiratory flora. finally, a last interesting finding of the study is that prevotella-associated profiles were similar to those of children with pathogen-dominated microbiota, and this could mean that even the microbiota in some cases contributes to inflammation in these patients. this could explain why some children with chronic cough and lower inflammation without respiratory species detected still respond to antibiotic treatment ( ) . nevertheless, further studies are needed to establish whether a high relative load of prevotella could explain the need of longer antibiotics ( weeks) among children with pbb ( , ) . it is therefore important to understand the role of the microbiota in the pathogenesis of pbbs to identify any other bacteria involved in the recurrence and progression to bronchiectasis ( ) . a biofilm is "an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix; bacterial growth and activity are substantially enhanced by the incorporation of a surface these organisms could tie to" ( ) . this matrix decreases antibiotic penetration protecting bacteria against antibiotics ( ) . it is reasonable to assume that a chronic bacterial bronchitis develops when one or more pathogens aggregate to form biofilms within the conducting airways dominating a niche. the prevalence of a single species or mixed populations within the biofilms drives a chronic inflammatory state, which induces a favorable environment for some bacteria such as non-typeable h. influenzae (nthi) ( ) . viral infection appears not only to enable the starting of both the surface attachment and of biofilms but also to be the trigger for the exacerbations characterized by the release of planktonic organism, which will generate an enhanced inflammatory response ( ) . the presence of biofilms can cause the need for prolonged antibiotic therapy, and it has been detected both in bal of children with bronchiectasis and with pbb ( ). different types of virus have been detected in bal from children with pbb, but the clinical significance of this is unclear. in a first study on these subjects, high virus detection was reported in pbb patients ( %) compared to controls ( %), and the most common identified virus was adenovirus (adv) in the pbb-bal ( %) compared with controls ( %) frequently encountered with h. influenza coinfection ( ) . the same study found other viruses such as rhinovirus ( %), human bocavirus ( %), and human coronavirus ( %) with overlapping prevalence between the two groups ( ) . there is only one study in the literature on this topic, published in , whose data are opposed to those already known. this study looks for common viruses in the bal of patients with pbb and controls. the detection rate is almost the same within the two groups ( . - . %) ( ) . moreover, no adv in bal in pbb cases were detected, in contrast to previous studies ( , ) . based on this conflicting data, nowadays, we can say that there is no certain evidence that pbb may be virus induced. the correlation between major airway injuries and recurrent bronchitis is well-known ( ) . airway malacia is detected frequently in children with pbb ( ) . it may affect airway clearance, therefore predisposing to pbb, although also airway inflammation may predispose to malacia in a vicious circle ( , ) . kompare et al., in their retrospective study about pbb and tracheobronchomalacia, have assessed children ( female and male) with protracted cough, wheeze, and/or noisy breathing in whom bal found ≥ cfu/ml of potentially pathogenic bacteria; children with other major conditions were excluded (asthma, cystic fibrosis, and other known chronic diseases) ( ) . they reported malacia in % of pbb cases ( ) . a prospective study by wurzel et al. on a cohort of children with pbb evidenced a correlation with tracheo-and/or bronchomalacia in % of ( ) . furthermore, airway malacia can both decrease effectiveness of cough and interfere with normal mucous movement, a crucial mechanism for clearing bacteria from the airways ( ) . as postulated by donnelly et al., airway malacia could induce an impairment of normal pulmonary defense mechanisms promoting the development of chronic cough and pbb ( ) . children with pbb usually do not have immunodeficiencies; therefore, most of them have normal serum immunoglobulin levels by age (igg, iga, igm, and ige) as well as normal antibodymediated response to protein (tetanus) and conjugated proteinpolysaccharide (h. influenzae type b) ( , ) . nevertheless, several studies report the presence of intense airway neutrophilia with a neutrophil percentage between . and %; no eosinophilia was found, and just one study reported percentage increase in lymphocytes ( , , , ) . lymphocyte subsets were normal, except for increased cd and cd natural killer cell level for age, probably associated with recent viral infection ( , , ) . increased levels of interleukin (il)- , il- β, and active matrix metalloproteinase- seems to correlate with the degree of neutrophilia ( ) . a study of chang et al. detected increased human β-defensin- and mannose-binding lectin levels, while activated caspase- -dependent proinflammatory pathways in response to nthi were also identified in pediatric patients with pbb, because both the innate pathogen recognition and clearance mechanisms were normal ( ) . in addition, higher levels of toll-like-receptor (tlr- ) and toll-like-receptor (tlr- ) in the bal of children with pbb are reported compared to controls ( ) . finally, another study focuses on the possibility of an impaired clearance of apoptotic cells by alveolar macrophages (efferocytosis). the remaining apoptotic cells may undergo secondary necrosis with proinflammatory effect, thus increasing chronic inflammation and tissue damage ( , ) . in , chang et al. proposed a paradigm where "pbb, csld and bronchiectasis shared common underlying pathobiological mechanisms and progressed variably along an increasing spectrum of severity" ( ) . the similarities are chronic wet cough, rattling breathing, defective mucociliary clearance, endobronchial bacterial infection, and neutrophilic airway inflammation ( ) . the major differences between these conditions consist in the clinical severity, the improvement to - weeks of adequate antibiotic treatment, chest high-resolution ct scan findings, and subsequent management ( , ) . starting from these observations, analysis were made to address the possible existence of more elements predicting the evolution of pbb in bronchiectasis capable of explaining why among children with recurrent pbb, some subjects do not show pulmonary sequelae, while other children develop bronchiectasis. a recent prospective longitudinal cohort study assessed the year outcomes of pediatric patients with pbb and detected two main risk factors for bronchiectasis: recurrent pbb (> episodes/years) and a positive bal culture for h. influenzae. this finding correlated with a higher risk of bronchiectasis (more than seven times) compared with no infection ( ) . moreover, authors showed that ∼ of children with pbb are diagnosed with bronchiectasis at years follow-up, with many experiencing recurrent episodes of pbb. this study provides further evidence to support a link between pbb and bronchiectasis in young children. this may also suggest the need to monitor children with pbb over time and to consider chest ct imaging in those with risk factors for bronchiectasis ( ) . noteworthy are some evidence showing that lower airways of pbb and bronchiectasis are characterized by marked neutrophilic inflammation with intense proinflammatory mediator responses such as interleukin- , matrix metalloproteinase- , and il- β. all these findings significantly differ to controls and support the hypothesis that lower airway microbiology and pathobiological aspects are similar in pbb and bronchiectasis ( , ) . according to the chest guideline and expert panel report, there is high-quality evidence that the administration of appropriate antibiotics among children aged ≤ years with wet/productive cough improves cough resolution, although further investigation should be undertaken when specific cough pointers (e.g., digital clubbing) are present. when the wet cough does not improve in response to weeks of antibiotic therapy, there is moderate-quality evidence that further investigations such as flexible bronchoscopy, chest ct scan, and immunity tests should be considered to look for an underlying disease ( ) . children with pbb should be treated with antibiotic for at least weeks. several studies have been performed, and use of prolonged antibiotic treatment has been shown to facilitate cough resolution compared to placebo ( ) . specifically in a randomized controlled trial conducted by marchant et al. ( ) , including children (median age, . years) with chronic wet cough (> weeks) a -week treatment with amoxicillin-clavulanate acid allowed cough resolution compared with placebo ( vs. %) ( ) . amoxicillin-clavulanate acid is the most commonly used antibiotic due to its activity against βlactamase, although other options such as oral second or third generation cephalosporins, trimethoprim-sulfamethoxazole, or a macrolide may be used among patients with ige-mediated reaction to penicillin ( ). nevertheless, oral cephalosporins because of its similarity to penicillins (e.g., ampicillin and cefalexin or cefaclor) should be avoided among these patients ( ) . some children require up to weeks of treatment. marchant j. et al. in their randomized controlled trial (rct) cited above shows that many of the children not responding after weeks of treatment had underlying tracheobronchomalacia ( ) , even though better evidence is needed to determine whether a prolonged course of antibiotics is beneficial, due to the inherent risk of antibiotic therapy. furthermore, during a different study on children eligible upon defined criteria [presence of chronic wet cough > weeks and having completed at least weeks of oral antibiotics directed against likely respiratory bacterial pathogens associated with pbb, cystic lung diseases (clds), and bronchiectasis] goyal v. et al. showed that children affected by chronic wet cough not improving after weeks of appropriate treatment have increased likelihood ( / , . %) of bronchiectasis on a chest ct scan ( ) . some clinicians prefer to use prolonged therapies even beyond the resolution of symptoms, and the rationale is that protecting the airways against the common respiratory bacteria for a longer period reduces the risk of reoccurrence and recovers airways integrity ( ). however, prolonged antibiotic treatments may cause dysbiosis and the selection of antibiotic-resistant strains ( ) . lastly, the role of weekly azithromycin in pbb is not clear even though it seems halving the rate of exacerbations in children with either csld or bronchiectasis ( , ) . bts cough guidelines suggest that all children with pbb should receive both - weeks of antibiotics ( ) and physiotherapy. pbb is a common cause of persistent wet cough in preschool children worldwide; it is frequently underdiagnosed or mistaken for other diseases such as postviral cough or asthma and therefore inadequately treated. considering pbb in the differential diagnosis of chronic wet cough in children allows an early and adequate antibiotic treatment to eradicate the infection. bal and antibiogram generally are not required, as well as chest x-ray, and clinical diagnosis is enough to start an empiric weeks therapy with amoxicillin-clavulanate acid. this therapy is mostly effective against the bacterial species involved, such as h. influenzae (nthi), s. pneumoniae, and m. catarrhalis. moreover, an adequate therapy prevents the onset of a prolonged inflammatory process potentially associated with structural damage of the lower airways, which may be involved in bronchiectasis. several studies provide evidences to support a link between pbb and bronchiectasis in children, and this emphasizes the necessity to consider this possibility of evolution and to carry out some more detailed investigations in the case of a clinical suspicion. ers statement on protracted bacterial bronchitis in children guidelines for evaluating chronic cough in pediatrics management of children with chronic wet cough and protracted bacterial bronchitis: chest guideline and expert panel report ers guidelines on the diagnosis and treatment of chronic cough in adults and children recommendations for the assessment and management of cough in children minimally important change in a parent-proxy quality-of-life questionnaire for pediatric chronic cough update on pediatric cough chronic wet cough: protracted bronchitis, chronic suppurative lung disease and bronchiectasis evaluation and outcome of young children with chronic cough protracted bacterial bronchitis: the last decade and the road ahead a multicenter study on chronic cough in children: burden and etiologies based on a standardized management pathway evaluation of children with chronic cough accompanied by a new clinical algorithm prospective characterization of protracted bacterial bronchitis in children how cigarette smoke skews immune responses to promote infection, lung disease and cancer utility of signs and symptoms of chronic cough in predicting specific cause in children an underestimated cause of chronic cough: the protracted bacterial bronchitis bronchoalveolar lavage in children comparison of cell profiles in separately evaluated fractions of bronchoalveolar lavage (bal) fluid in children protracted bacterial bronchitis: bronchial aspirate versus bronchoalveolar lavage findings: a single-centre retrospective study wet cough in children: infective and inflammatory characteristics in broncho-alveolar lavage fluid outcomes in children treated for persistent bacterial bronchitis three clinically distinct chronic pediatric airway infections share a common core microbiota the microbiota in bronchoalveolar lavage from young children with chronic lung disease includes taxa present in both the oropharynx and nasopharynx the impact of persistent bacterial bronchitis on the pulmonary microbiome of children multiple respiratory microbiota profiles are associated with lower airway inflammation in children with protracted bacterial bronchitis randomised controlled trial of amoxycillin clavulanate in children with chronic wet cough biofilms: microbial life on surfaces mechanisms of antibiotic resistance in bacterial biofilms persistent and recurrent bacterial bronchitis-a paradigm shift in our understanding of chronic respiratory disease. front pediatr detecting respiratory viruses in children with protracted bacterial bronchitis tracheomalacia and bronchomalacia in children: incidence and patient characteristics bronchoscopic findings in children with non-cystic fibrosis chronic suppurative lung disease protracted bacterial bronchitis in young children: association with airway malacia airway mucus function and dysfunction mediators of neutrophil function in children with protracted bacterial bronchitis pulmonary innate immunity in children with protracted bacterial bronchitis clinical characteristics of protracted bacterial bronchitis in chinese infants prospective assessment of protracted bacterial bronchitis: airway inflammation and innate immune activation burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease protracted bacterial bronchitis is a precursor for bronchiectasis in children: myth or maxim? breathe protracted bacterial bronchitis in children: natural history and risk factors for bronchiectasis children with chronic wet or productive cough-treatment and investigations does failed chronic wet cough response to antibiotics predict bronchiectasis? the intestinal microbiome in early life: health and disease long-term azithromycin for indigenous children with non-cysticfibrosis bronchiectasis or chronic suppurative lung disease (bronchiectasis intervention study): a multicentre, double-blind, randomised controlled trial mp and mg performed the literature review. gr coordinated the writing group. all authors critically reviewed the manuscript and read and approved the final version. key: cord- -kahi cbc authors: miller, robert f.; lipman, marc c.i. title: pulmonary infections date: - - journal: clinical respiratory medicine doi: . /b - - . - sha: doc_id: cord_uid: kahi cbc nan it is hard to believe that the first consistent reports of acquired immunodeficiency syndrome (aids) were only years ago. since then, respiratory and general physicians have become accustomed to dealing with an extraordinary range of esoteric and previously unheard of conditions. pneumocystis carinii (now known as pneumocystis jirovecii) pneumonia is frequently part of the differential diagnosis of treatment-nonresponsive pneumonic illness. human immunodeficiency virus (hiv) testing is almost routinely offered as a "rule out" test in clinical cases that defy simple diagnosis. in many parts of the developing world, advanced hiv disease is unfortunately the likeliest reason for seeking medical care. the change this has wrought on people, countries and their economies is huge and depressing. the isolation of hiv from patients with aids in the mid s paved the way for intensive research with the ultimate development of drugs directed against this chronic infection. however, despite such advances, the methods by which hiv infection leads to severe immune dysregulation and clinical disease are still not fully defined. the introduction in , in the developed world, of highly active antiretroviral therapy, also known as combination antiretroviral therapy (cart) hereto referred to as haart, has altered the natural history of this extraordinary condition. before haart, defined as a combination of medications that usually includes at least three potent anti-hiv agents, treatment largely consisted of specific opportunistic infection management and less effective antiretroviral therapy. the clinical consequences of this change are enormous. the relative hazard for development of pneumocystis pneumonia (pcp) in an hivinfected individual has fallen by more than %. drug therapy does have a down side. it has significant unwanted effects, as well as major interactions with other medication (e.g., rifamycins used in treating tuberculosis). the profound change in immunity induced by haart may also lead to disease (the immune reconstitution inflammatory syndrome [iris] ). notwithstanding haart, respiratory disease remains an important cause of morbidity and mortality. much of the world cannot afford such medication, and more than two thirds of hiv-infected individuals have at least one respiratory episode during the course of their illness. in the early stages of hiv infection, when patients have relatively preserved immune responses, individuals have the same infections found in the general population, although at a greater incidence. with progressive hiv disease, subjects are at an increased risk of opportunistic disease. for example, the north american prospective study of pulmonary complications of hiv infection (pspc), a multicenter cohort drawn from all hiv risk groups at various stages of immunosuppression, revealed over an -month study period that of approximately subjects who were not using haart: % reported an upper respiratory tract infection % had an episode of acute bronchitis % had acute sinusitis % had bacterial pneumonia % had pcp develop the immune dysregulation that arises from hiv infection means that bacteria, mycobacteria, fungi, viruses, and protozoa can all cause disease in subjects with advanced infection. table - shows the organisms that typically infect the lung in hiv disease. of these, bacterial infections, tuberculosis, and pcp are the most important. in the west, % of aids diagnoses are due to pcp. this chapter provides a brief general overview of the epidemiology and pathogenesis of hiv infection before concentrating on hiv and its infectious pulmonary complications. it is reported that by the end of , . million individuals worldwide had acquired hiv infection (figure - ). of these, more than % are thought to have had aids develop (for definition of aids, see tables - and - , and box - ). globally, . million individuals acquired hiv infection in , and over this time . million died of aids. the developing world has been most affected. sub-saharan africa is the current epicenter of the pandemic (two thirds of all infections); here, one in five adults is hiv infected. south and southeast asia are responsible for almost a fifth of the estimated hiv global burden. in central-eastern europe and central asia, there are currently . million hivinfected individuals. in the developed world, north america and western europe account for approximately . million and , infections, respectively. most of these are spread through sexual contact, although vertical (mother-to-child) and bloodborne infections are also common. in the developing world, heterosexual transmission is the norm; however, in north america and europe, homosexual and bisexual men constitute the largest group of infected individuals. hiv was first isolated in from patients with symptoms and signs of immune dysfunction. two subtypes (hiv- and hiv- ) have subsequently been identified. hiv- (hereafter referred to as hiv) is responsible for most infections, has a more aggressive clinical course, and is the focus of this chapter. hiv is a human retrovirus belonging to the lentivirus family. cell-free or cell-associated hiv infects through attachment of its viral envelope protein (gp ) to the cd antigen complex on host cells. the cd receptor is found on several cell types, although the t-helper lymphocyte is the main site of hiv infection in the body. hiv gp must also bind to a cell surface protein coreceptor called chemokine receptor (ccr ) or to other co-receptors, including cxcr , depending on the host cell type. polymorphisms in genes for ccr may affect disease progression by reducing the ability of hiv to enter and infect cells. however, at a population level, this effect is small. once hiv is inside the cell, it can, by use of the enzyme reverse transcriptase (rna-dependent dna polymerase), transcribe its hiv rna into a dna copy that can translocate to the nucleus and integrate with host cell dna by use of its viral integrase. the virus (as proviral dna) remains latent in many cells until the cell itself becomes activated. this may arise from cytokine or antigen stimulation. the viral genetic material is then transcribed into new rna, which, in the form of a newly created virion, buds from the cell surface and is free to infect other cd -bearing cells. hiv infection directly attacks the immune system and in particular the t-helper cells that are central to a coordinated immune response. this leads to progressive immune dysfunction and an inability to resist opportunistic disease. the pathogenic process is not well defined, although it is thought that at the time of primary infection, hiv spreads to the lymph nodes, circulating immune cells, and thymus. this is a massive viral infection of the human host; and although there seems to be a relatively potent immune response, in fact, this initial onslaught is so devastating (targeting as it does specific memory t cells responsible for sustaining long-term protective immunity) that the scene is set for progressive immune failure. this occurs through a combination of direct cell killing by hiv replicating within cells, as well as the negative effects of chronic immune activation. ultimately, these lead in most individuals to immune system destruction and dysfunction. this is reflected not only by clinical disease indicating profound immunosuppression but also by a measurable reduction in the circulating absolute cd cell count, the percentage of t cells expressing cd markers, and in the progressive reduction in cd /cd t-cell ratio. the use of haart, as well as preventative (prophylactic) therapies for opportunistic infections, has changed the clinical picture of hiv disease in countries where these interventions are available. death rates have fallen to one sixth of their previous levels. however, in the absence of such treatments, the median interval between hiv seroconversion and progression to aids in the developed world has been estimated to be years, although rather less in resource-poor countries. almost all individuals have aids develop if untreated, and without haart, % of these will die within years. in many parts of the world, the main causes of death in patients with hiv infection include bacterial pneumonia, tuberculosis, and pcp. the course of hiv infection can be divided clinically into several distinct periods: acquisition of the virus seroconversion, with or without a clinical illness (primary hiv infection) clinically silent period, lasting several months to years development of symptoms and signs indicating some degree of immunosuppression aids (where the subject has opportunistic disease implying profound immunosuppression [e.g., pcp]) the time from acquisition of hiv infection to development of detectable antibodies (the "window" period) is usually approximately - weeks. between and % of individuals who become infected will have a seroconversion illness. hiv antibody is normally present within - weeks of these symptoms, although this can take longer. hiv rna in peripheral blood is detectable before this and is often used to confirm infection. the nonspecific features of primary hiv infection are almost always self-limiting, and typically seroconversion mimics a "flulike" illness or glandular fever. most individuals with primary hiv infection recover from the acute symptoms within weeks. a proportion may have persistent symmetric generalized lymphadenopathy. there is no difference in prognosis in this group compared with asymptomatic hiv-positive individuals. although a proportion of individuals remain completely well without any treatment for an extended period (approximately % after years), many hiv-infected individuals have minor symptoms and signs suggesting immune dysfunction. examples of these include new or worsening rashes (including herpes simplex), tiredness, cough, and low-grade anemia. certain clinical symptoms and signs provide important prognostic information. most studies have shown that oral candidiasis and constitutional symptoms (e.g., malaise, idiopathic fever, night sweats, diarrhea, and weight loss) are the strongest clinical predictors of progression to aids. the term aids was created as an epidemiologic tool to capture those conditions that early in the hiv epidemic seemed to suggest significant immune destruction. over time, it has been modified to incorporate the expanding spectrum of recognized diseases affecting immunosuppressed individuals, such as cervical carcinoma and recurrent bacterial pneumonia (see box - ). the centers for disease control and prevention (cdc) classification included an immunologic criterion for aids (cd count < cells/ml or cd percentage < % of total lymphocytes) regardless of clinical symptoms (see table - ). these data are used to define a point at which the risk of severe opportunistic infection rises dramatically. an example of this can be seen in the multicenter aids cohort study (macs) of homosexual and bisexual men without aids, which found that the incidence of pcp in subjects who did not use prophylaxis rose from . % at months in men with a baseline cd count > cells/ml to . % in those with a cd count < cells/ml. apart from cervical carcinoma, aids indicator diseases differ little between men and women. injecting drug users have a high incidence of wasting syndrome, recurrent bacterial pneumonia, and tuberculosis. geographic differences in diseases occur that reflect the opportunistic pathogens present in the local environment (e.g., histoplasmosis or visceral leishmaniasis usually occur only in patients from endemic areas). in the developed world, sexual, racial, and hiv risk factor survival differences after an aids diagnosis mainly arise from variation in ease of access to medical care. it is certainly the case, however, that better treatment outcomes are associated with genuine specialist care provided by people with extensive experience in the field. in countries where haart is available, the spectrum of hiv-related disease has changed. in the eurosida cohort (a pan-european prospective study of hiv infection), between and , opportunistic infections associated with very low cd counts (e.g., cytomegalovirus [cmv] retinitis and mycobacterium avium-intracellulare complex [mac] infection) were observed less frequently over time. malignant disease, such as non-hodgkin lymphoma, increased as an aids-defining event between and . although death rates have fallen in haart-treated populations, there has been a rise in the proportion of non-aids deaths. in some series, this accounts for most events. causes include liver disease (often caused by viral hepatitis) and cancer, as well as cardiovascular disease and drug-related toxicity. in such circumstances, aids deaths usually occur among patients who have not accessed medical care regularly and who are initially seen with advanced hiv disease. a new manifestation of opportunistic infection has been described in patients commencing haart. the immune reconstitution disease, iris, may cause severe, if temporary, clinical illness as the individual's immunity recovers. patients will appear to develop a relapse of their original (and partially treated) disease. this is often seen in mac infection, tuberculosis, hepatitis b, cmv retinitis, and herpesviral infection. metabolic complications of haart, such as ischemic heart disease and diabetes, are a potential problem in hiv practice in the developed world. a significant number of individuals taking haart also experience drug toxicity. an increasing number of patients are also surviving to manifest symptoms associated with chronic hepatitis b and c infection. hivassociated nephropathy (often with chronic kidney disease) is common in black africans and is a significant cause of long-term morbidity. laboratory markers and clinical symptoms (e.g., oral thrush) can independently reflect the immune changes that lead to serious disease. staging systems have been developed that can predict the risk of progression to severe opportunistic disease (aids). the fall in absolute blood cd t-lymphocyte count is the most widely used prognostic marker, although cd counts may be affected by a number of factors apart from hiv, including intercurrent infection, cigarette smoking, exercise, time of day, and laboratory variation. the percentage of cd cells and ratio of cd /cd cells are more stable measures and may be used if the cd absolute counts seem to vary widely from visit to visit. measurement of plasma hiv rna "viral load" provides important prognostic information that can both guide therapy and suggest long-term outcome. it has a particular value in subjects who are clinically well and have high cd counts, because it can give some indication of the expected speed of clinical progression. it is clear from the frequency with which hiv-related respiratory disease occurs that the pulmonary immune response is profoundly dysregulated. however, the mechanism underlying this has not been fully explained. in part, this is because the alterations that arise reflect the complex interplay between systemically derived hiv and other circulating antigens trafficking through the pulmonary vasculature, local immune cells, and airborne antigen. most studies investigating the pulmonary immune response have used in vitro cell culture systems that seek to mimic the pulmonary environment or in some cases bronchoscopy and bronchoalveolar lavage (bal) to recover lung cells from infected individuals. until recently, these have been performed on symptomatic patients who required bronchoscopy as a diagnostic procedure. such subjects generally have advanced hiv infection, are often taking a number of different drugs (including antiretrovirals), and may have any number of different pathogens causing their pulmonary disease-which by themselves can influence the immune findings. finally, the question of whether bal fluid truly represents the site of the immune response (the lung parenchyma) remains unclear. for all these reasons, reported data should be interpreted with some care. an individual's risk for respiratory disease is determined by his or her medical history (e.g., receipt of effective preventative or antiretroviral therapy), place of residence and travel history (e.g., the influence of geography on mycobacterial and fungal disease), and state of host immunity. falling blood cd counts or high plasma rna "viral loads" increase the chance of respiratory infection, with an increased spectrum of potential organisms responsible for infection in the more immunosuppressed individual. for example, hiv-infected individuals with a cd count < cells/ml are four times more likely to have one episode of bacterial pneumonia per year than those with higher cd cell counts. more exotic organisms are found in subjects with very low cd counts. these include bacteria such as rhodococcus equi and nocardia asteroides and fungi such as aspergillus species and penicillium marneffei. just as with p. jirovecii, this reflects the importance of t-cell depletion and macrophage dysfunction in the loss of host immunity (a process that has been confirmed by animal experiments). among hiv-infected patients, injecting drug users are at greatest risk for development of bacterial pneumonia and tuberculosis. individuals who have had previous respiratory episodes (pcp or bacterial pneumonia) seem to be at increased risk of further disease. whether this relates to host or environmental factors is not certain, although it seems likely that structural lung damage and abnormal pulmonary physiology would, in part, contribute to this. this argument is supported by the increased rates of pneumonia in hiv-infected smokers compared with nonsmokers. recent work has shown chronic obstructive pulmonary disease (copd) and lung cancer occur more frequently among hiv-infected individuals compared with the general population. given that a large number of hiv-infected individuals smoke heavily, there is a pressing need to target this population for smoking cessation. this is reinforced by the association demonstrated in some (but not all) studies between smoking and a more rapid progression to first aids illness and death. the presentation mimics bacterial exacerbations of chronic obstructive lung disease; most patients have a productive cough and fever. the pathogens commonly identified are similar to those in the general population (i.e., streptococcus pneumoniae and haemophilus influenzae). however, patients with advanced disease may be infected with pseudomonas aeruginosa or staphylococcus aureus. response to appropriate antibiotic therapy in conventional doses is good, although relapses frequently occur. bronchiectasis is increasingly recognized in hiv-infected patients with advanced hiv disease and low cd lymphocyte counts. it probably arises secondary to recurrent bacterial or p. jirovecii infections. the diagnosis is most often made by high-resolution/fine-cut computed tomography (ct) scanning. its prevalence has not been accurately determined, although with improved survival from both opportunistic infections and hiv disease, it is likely that it will be increasingly common in clinical practice. the pathogens isolated in patients with bronchiectasis are those seen in bronchitis. in addition, pseudomonas cepacia and moraxella catarrhalis have been described. community-acquired bacterial pneumonia occurs more frequently in hiv-infected patients than in the general population. it is especially common in hiv-infected injecting drug users. the spectrum of bacterial pathogens is similar to that in non-hiv-infected individuals (see table - ). s. pneumoniae is the most common cause, followed by h. influenzae. hiv-infected individuals with s. pneumoniae pneumonia are frequently bacteremic. in one study, the rate of pneumococcal bacteremia in hiv-infected individuals was times that of an hiv-negative population. more recent work has confirmed this to be the case for all causes of hiv-related bacterial pneumonia. typically, blood cultures have a -fold increased pickup rate in hiv-positive patients. the widespread use of haart has led to some decrease in rates of bacterial pneumonia and bacteremia, although they are still considerably higher than those seen in a non-hiv-infected population. bacterial pneumonia has a similar presentation in hivinfected and uninfected individuals. chest radiographs are frequently atypical, mimicking pcp in up to % of cases ( figure - ). by contrast, radiographic lobar or segmental consolidation may also be seen in a wide range of bacterial organisms (figure - ); these include s. pneumoniae, p. aeruginosa, h. influenzae, and mycobacterium tuberculosis. pcp may also present with lobar or segmental consolidation. in subjects with more advanced hiv disease and low cd lymphocyte counts, p. aeruginosa and s. aureus also cause pneumonia. complications of bacterial pneumonia frequently occur, and pleural effusions are twice as likely in hiv infection (often occurring with s. aureus infection); empyema and intrapulmonary abscess formation are present in up to % of patients. inevitably, the mortality rate is high (approximately %). nocardia asteroides infection. this has been reported in patients with advanced hiv disease and low cd lymphocyte counts. the widespread use of trimethoprim/sulfamethoxazole (tmp/smx) for prophylaxis of pcp may have reduced the incidence of infection. the clinical presentation is often indistinguishable from that of other bacterial infections. chest radiographic appearances may mimic tuberculosis (see later), with upper lobe consolidation, cavitation, interstitial infiltrates, pleural effusion, and hilar lymphadenopathy. the diagnosis is made by identification of the organism in sputum/bal fluid or lung tissue. rhodococcus equi. r. equi usually produces pneumonia in patients who have advanced hiv infection and have been in contact with farm animals or with soil from fields or barns where animals are housed. the presentation is subacute, with - weeks of cough, dyspnea, fever, and pleuritic chest pain. the chest radiograph typically shows consolidation with cavitation. pleural effusions are common. the diagnosis is usually made by culture of sputum or blood; bronchoscopy with bal or pleural aspiration may be necessary in some cases. bartonella henselae. b. henselae is a gram-negative bacillus that causes bacillary angiomatosis in hiv-infected patients. clinically, the cutaneous lesions may mimic kaposi sarcoma, from which they may be distinguished by demonstration of organisms in tissue with warthin-starry silver stain. bacillary angiomatosis may also infect the lungs, where it produces endobronchial red or violet polypoid angiomatous lesions, which may resemble kaposi sarcoma. biopsy is necessary to confirm a diagnosis. tuberculosis hiv infection is associated with at least a -fold increased risk of an individual having active tuberculosis develop compared with noninfected subjects. taken together with its ability to infect both the immunosuppressed and immunocompetent, tuberculosis is perhaps, therefore, the single most important disease associated with hiv infection. it is estimated that there are at least million individuals with hiv-tuberculosis coinfection. as such, tuberculosis is a major cause of hiv-related morbidity and mortality. it is also a major driver in both resource-rich and resource-poor countries for the current overall increase in tuberculosis rates. where hiv infection is endemic, tuberculosis control at a population level is almost impossible if treatment for both infections is not available. in the united kingdom, many centers routinely offer hiv antibody testing to all patients with tuberculosis, regardless of risk factors for hiv infection. in the united states, the cdc now recommends hiv testing as a routine part of health care for all patients aged - accessing medical services. the advantage of this is that individuals who are found to be hiv infected can be given haart. furthermore, strategies to modify high-risk behavior and reduce ongoing hiv transmission may be offered. active tuberculosis can occur at any stage of hiv infection, and unlike almost every other hiv-related infection, may do so despite effective antiretroviral therapy. in the united states, united kingdom, and most european countries, reporting of tuberculosis in both hiv-infected and non-hiv-infected individuals is mandatory. clinical disease in hiv-infected patients may arise in several different ways: by reactivation of latent tuberculosis, by rapid progression of pulmonary infection, and by reinfection from an exogenous source. pulmonary disease is the most common presentation; and clinical manifestations are related to the level of an individual's cell-mediated immunity. for example, subjects with early hiv disease have clinical features similar to "normal" adult postprimary disease (table - ). symptoms typically include weight loss, fever with sweats, cough, sputum, dyspnea, hemoptysis, and chest pain. these patients may have no clinical features to suggest associated hiv infection. the chest radiograph frequently shows upper lobe consolidation, and cavitary change is common (figure - ) . the tuberculin skin test (purified protein derivative [ppd] ) is usually positive, and the likelihood of spontaneously expectorated sputum or bal fluid being smear positive for acid-fast bacilli is high. in individuals with advanced hiv disease (i.e., low cd lymphocyte counts and clinically apparent immunosuppression), it may be difficult to diagnose tuberculosis. the clinical presentation here is often with nonspecific symptoms. fever, weight loss, fatigue, and malaise may be mistakenly ascribed to hiv infection itself. in this context, pulmonary tuberculosis is often similar to primary infection, with the chest radiograph showing diffuse or military-type shadowing ( figure - ), hilar or mediastinal lymphadenopathy, or pleural effusion; cavitation is unusual. in up to % of patients the chest radiograph may appear normal; in others, the pulmonary infiltrate can be bilateral, diffuse, and interstitial in pattern, thus mimicking pcp. hilar lymphadenopathy and pleural effusion may also be produced by pulmonary kaposi sarcoma or lymphoma, with which m. tuberculosis may coexist. the tuberculin skin test is usually negative, and spontaneously expectorated sputum and bal fluid are often smear negative (but culture positive). in addition to pulmonary tuberculosis, extrapulmonary disease occurs in a high proportion of hiv-infected individuals with low cd lymphocyte counts (< cells/ml). mycobacteremia and lymph node infection ( figure - ) are common, but involvement of bone marrow, liver, pericardium, meninges, and brain also occurs. evidence of extrapulmonary tuberculosis should be sought in any hiv-infected patient with suspected or confirmed pulmonary tuberculosis, by culture of stool, urine, and blood or bone marrow. traditional solid phase culture and speciation techniques may take - weeks. liquid culture methods (e.g., bactec, becton dickinson) that detect early growth may provide a diagnosis in only - weeks. molecular diagnostic tests that use m. tuberculosis genome detection (e.g., by polymerase chain reaction [pcr] ) offer the possibility of yet more rapid diagnosis (within hours), but are not yet in routine clinical use. they are also less useful in primary samples with low bacterial load (e.g., smear negative sputum)-which is often when they will be most needed in hiv-coinfected patients. the recent description of simple, but highly sensitive and specific, methods that use the inoculation of large quantities of, for example, sputum onto microscopic plates with subsequent rapid detection (in days) of both mycobacterial growth and resistance patterns (mods) is of great potential significance. until the results of culture and speciation are known, acidfast bacilli identified in respiratory samples, biopsy tissue, an aspirate, or blood in an hiv-infected individual, regardless of the cd lymphocyte count, should be regarded as being m. tuberculosis, and conventional antituberculosis therapy should be commenced. if culture fails to demonstrate m. tuberculosis and instead another mycobacterium (see later) is identified, treatment can be modified. multiple drug-resistant (mdr) tuberculosis-that is, m. tuberculosis that is resistant to isoniazid and rifampicin (rifampin), with or without other drugs, is now an important clinical problem in hiv-infected individuals in the united states, where it is responsible for approximately % of all tuberculosis in hiv-infected patients. outbreaks of mdr tuberculosis have occurred in both hiv-infected and non-hiv-infected individuals in the united states in prison facilities, hostels, and hospitals. similar outbreaks have also been documented among hiv-infected patients in europe. inadequate treatment (including case management and supervision of medication) of tuberculosis and poor patient compliance with antituberculosis therapy are the most important risk factors for development of mdr tuberculosis. other cases have arisen because of exogenous reinfection of profoundly immunosuppressed hiv-infected patients who are already receiving treatment for drug-sensitive disease. despite antituberculosis therapy, the median survival in hiv-infected individuals with mdr-tuberculosis was initially only - months. recently this has improved, largely because of an increased awareness of the condition with early initiation of suitable therapy as determined by drug sensitivity testing. extensively drug-resistant (xdr) tuberculosis-that is, m. tuberculosis resistant to isoniazid and rifampicin (rifampin), plus any fluoroquinolone and one or more of the three injectable second-line drugs (capreomycin, kanamycin and amikacin)-is an increasingly important clinical problem. originally described in south africa in association with hiv infection, xdr tuberculosis has also been identified in most parts of the world. as of march , countries had reported at least one case; although in many places, testing for fluoroquinolone sensitivity is not standard practice; this number may be, in fact, a huge underestimate. what is of concern about xdr tuberculosis is that, despite specific therapy, mortality is high among hiv-infected individuals. the current picture seems to mirror early reports of mdr tuberculosis in hiv infection: in the original south african study from kwazulu natal, survival was less than weeks from the time of receipt of the first sputum sample. mycobacterium avium-intracellulare complex. before the widespread availability of haart, disseminated mac infection developed in up to % of hiv-infected patients. it remains a problem in patients with advanced hiv disease not receiving antiretroviral therapy and who have cd lymphocyte counts < cells/ml. clinical presentation is nonspecific and may be confused with the effects of hiv itself. fever, night sweats, weight loss, anorexia, and malaise are common. anemia, hepatosplenomegaly, abdominal pain, and chronic diarrhea are frequent findings. the diagnosis of disseminated mac infection is based on culture of the organism from blood, bone marrow, lymph node, or liver biopsy specimens. also, mac is frequently identified in bal fluid, sputum, stool, and urine, but detection of the organism at these sites is not diagnostic of disseminated infection. evidence of pulmonary mac infection is not usually obtained from a chest radiograph, which may be negative or show nonspecific infiltrates. rarely, focal consolidation, nodular infiltrates, and apical cavitation (resembling m. tuberculosis) have been reported. mycobacterium kansasii. mycobacterium kansasii is the second most common nontuberculous opportunistic mycobacterial infection in hiv-infected individuals and usually appears late in the course of hiv infection in patients with cd lymphocyte counts < cells/ml. the most frequent presentation is with fever, cough, and dyspnea. in approximately two thirds of those who have m. kansasii infection, the disease is localized to the lungs; the remainder have disseminated disease that affects bone marrow, lymph node, skin, and lungs. the diagnosis is made by culture of the organism from respiratory secretions or from bone marrow, lymph node aspirate, or skin biopsy. focal upper lobe infiltrates with diffuse interstitial infiltrates are the most common radiographic abnormalities; thin-walled cavitary lesions and hilar adenopathy have also been reported. mycobacterium xenopi. mycobacterium xenopi may occasionally be isolated from sputum or bal fluid samples, but its significance is uncertain. patients have low cd counts, and m. xenopi is usually accompanied by isolation of a copathogen, such as p. jirovecii. treatment of the latter condition is associated in most cases with resolution of symptoms. there is some evidence that starting haart prevents disease recurrence, provided there is an adequate immune response. pneumocystis jirovecii pneumonia. the development of pcp is largely related to underlying states of immunosuppression induced by malignancy or treatment thereof, organ transplantation, or hiv infection. in in the united states, united kingdom, europe, and australasia, pcp is largely seen only in hiv-infected individuals unaware of their serostatus or in those who are intolerant of, or noncompliant with, anti-p. jirovecii prophylaxis and haart. until recently, p. jirovecii was regarded taxonomically as a protozoan, on the basis of its morphology and the lack of response to antifungal agents such as amphotericin b. the organism has now been ascribed to the fungal kingdom. the demonstration of antibodies against p. jirovecii in most healthy children/adults suggests that organisms are acquired in childhood and persist in the lungs in a dormant phase. subsequent immunosuppression (e.g., as a result of hiv infection) allows the fungus to propagate in the lung and cause clinical disease. however, this "latency" hypothesis is challenged by several observations: p. jirovecii cannot be identified in the lungs of immunocompetent individuals. "case clusters" of pcp in health care facilities suggest recent transmission. different genotypes of p. jirovecii are identified in each episode in hiv-infected patients who have recurrent pcp. genotypes of p. jirovecii in patients who have pcp correlate with place of diagnosis and not with their place of birthsuggesting infection has been recently acquired. taken together, these data suggest that pcp arises by reinfection from an exogenous source. the clinical presentation of pcp is nonspecific, with an onset of progressive exertional dyspnea over days or weeks, together with a dry cough with or without expectoration of minimal quantities of mucoid sputum. patients often complain of an inability to take a deep breath, which is not due to pleurisy (table - ) . fever is common, yet patients rarely complain of temperatures or sweats. in hiv-infected patients, the presentation is usually more insidious than in patients receiving immunosuppressive therapy, with a median time to diagnosis from onset of symptoms of more than weeks in those with hiv compared with less than week in non-hiv-infected patients. in a small proportion of hiv-positive individuals, the disease course of pcp is fulminant, with an interval of only - days between onset of symptoms and progression to development of respiratory failure. in others, it may be much more indolent, with respiratory symptoms that worsen almost imperceptibly over several months. rarely, pcp may present without respiratory symptoms as a fever of undetermined origin. clinical examination is usually remarkable only for the absence of physical signs; occasionally, fine, basal, end-inspiratory crackles are audible. features that would suggest an alternative diagnosis include a cough productive of purulent sputum or hemoptysis, chest pain (particularly pleural pain), and signs of focal consolidation or pleural effusion (see table - ). it should be noted that infection with more than one pathogen occurs in almost one fifth of individuals, and thus symptoms may be the product of several agents. the chest radiograph in pcp is typically unremarkable initially. later, diffuse reticular shadowing, especially in the perihilar regions, is seen and may progress to diffuse alveolar consolidation that resembles pulmonary edema if untreated or if the patient is seen late in disease. at this stage, the lung may be massively consolidated and almost airless (figure - ) . up to % of chest radiographs are atypical, showing lobar consolidation, honeycomb lung, multiple thin-walled cystic air space formation (pneumatoceles), intrapulmonary nodules, cavitary lesions, pneumothorax, and hilar and mediastinal lymphadenopathy. predominantly apical changes, resembling tuberculosis, may occur in patients who have pcp develop having received anti-p. jirovecii prophylaxis with nebulized pentamidine (figure - ). all these radiographic changes chest radiograph early: perihilar "haze" or bilateral interstitial shadowing late: alveolar-interstitial changes or "white out" (marked alveolar consolidation with sparing of apices and costophrenic angles) arterial blood gases pao : early, normal: late, low paco : early, normal or low; late, normal or high are nonspecific, and similar changes occur with other pulmonary pathogens, including pyogenic bacterial, mycobacterial, and fungal infection, as well as kaposi sarcoma and nonspecific interstitial pneumonitis. respiratory symptoms in an immunosuppressed, hiv-infected individual with a negative chest radiograph should not be discounted, because over an interval of - days radiographic abnormalities may appear. the diagnosis of pcp is made by demonstration of the organism in induced sputum, bal fluid, or lung biopsy material by use of histochemical or immunofluorescence techniques. the early promise of molecular diagnostic techniques has not been borne out. many fungal infections of the lung are confined to specific geographic regions, although with widespread travel, they may present in patients outside these areas. candida, aspergillus, and cryptococcus species are ubiquitous and occur worldwide. in contrast to infections of the oropharynx and esophagus, candidal infection of the trachea, bronchi, and lungs is rare in hiv-infected patients, as are candidemia, disseminated candidiasis, and deep focal candidiasis. the clinical presentation of pulmonary candidal infection has no specific features. chest radiography is equally nonspecific-it may be negative or show patchy infiltrates. isolation of candida from sputum may simply represent colonization and does not mean the patient has candidal pneumonia. indirect evidence may be obtained from positive cultures or rising antibody titers. however, in hivinfected patients, a high antibody titer alone is a less reliable indicator, and antibodies may be absent in proven cases of invasive candidal infection. some correlation occurs between identification of large quantities of candida species in bal fluid and candida species as the cause of pneumonia. definitive diagnosis is made by lung biopsy. by contrast with patients immunosuppressed and rendered neutropenic by systemic chemotherapy, infection with aspergillus species is relatively rare in hiv-positive individuals. risk factors for aspergillosis are neutropenia, which is commonly drug induced (zidovudine or ganciclovir), or patient's receipt of corticosteroids. fever, cough, and dyspnea are the most common presenting symptoms, but pleuritic chest pain and hemoptysis are found in approximately one third of patients. patterns of pulmonary disease include cavitating upper lobe disease, focal radiographic opacities resembling bacterial pneumonia, bilateral diffuse and patchy opacities (nodular or reticular-nodular in pattern), pseudomembranous aspergillosis, which may obstruct the lumen of airways, and tracheobronchitis. diagnosis of pulmonary aspergillosis is made by the identification of fungus in sputum, sputum casts, or bal fluid associated with respiratory tract tissue invasion ( figure - ). the role of antigen testing (such as galactomannan assays), which is commonplace in hematology patients at risk of invasive aspergillus, has not been clearly defined in hiv-infected individuals. infection may present in one of two ways: either as primary cryptococcosis or complicating cryptococcal meningitis as part of disseminated infection with cryptococcemia, pneumonia, and cutaneous disease (umbilicated papules mimicking molluscum contagiosum; figure - ). primary pulmonary cryptococcosis presents in a very nonspecific way and is frequently indistinguishable from other pulmonary infections. in disseminated infection, the presentation is frequently overshadowed by headache, fever, and malaise (caused by meningitis). the duration of onset may range from only a few days to several weeks. examination may reveal skin lesions, lymphadenopathy, and meningism. in the chest, signs may be absent or crackles may be audible. arterial blood gas tensions may be normal or show hypoxemia. the most common abnormality on the chest radiograph is focal or diffuse interstitial infiltrates. less frequently, masses, mediastinal or hilar lymphadenopathy, nodules, and effusion are noted. the diagnosis of cryptococcal pulmonary infection ( figure - ) is made by identification of cryptococcus neoformans (by staining with india ink or mucicarmine, and by culture) in sputum, bal fluid, pleural fluid, or lung biopsy. cryptococcal antigen may be detected in serum by use of the cryptococcal latex agglutination (crag) test. titers are usually high but may be negative in primary pulmonary cryptococcosis, in which case bal fluid (crag) is positive. in patients with disseminated infection, c. neoformans may also be cultured from blood and cerebrospinal fluid. the mortality rate is high in this disseminated form (up to %). the endemic mycoses caused by histoplasma capsulatum, coccidioides immitis, and blastomyces dermatitidis are found in hiv-infected patients living in north america (especially the mississippi and ohio river valleys). histoplasmosis is also found in southeast asia, the caribbean islands, and south america. coccidioidomycosis is endemic in the southwest united states (southern california), northern mexico, and in parts of argentina and brazil. blastomycosis has a similar distribution, with an extension north into canada. progressive, disseminated histoplasmosis in patients with hiv typically presents with a subacute onset of fever and weight loss; approximately % of patients have mild respiratory symptoms with a nonproductive cough and dyspnea. hepatosplenomegaly is frequently found on examination, and a rash (similar to that produced by cryptococcus species) may be seen. rarely, the presentation may be rapidly fulminant, with clinical features of the sepsis syndrome, including anemia or disseminated intravascular coagulation. the chest radiograph may be unremarkable (in up to one third of patients), although characteristic abnormalities are bilateral, widespread nodules - mm in size. other radiographic features are nonspecific and include interstitial infiltrates, reticular nodular shadowing, and alveolar consolidation. histoplasmosis may disseminate to the central nervous system and produce meningoencephalitis or mass lesions. the diagnosis is made reliably by identification of the organism in wright-stained peripheral blood or by giemsa staining of bone marrow, lymph node, skin, sputum, bal fluid, or lung tissue. it is important that identification is confirmed by detection of h. capsulatum var. capsulatum polysaccharide antigen by radioimmunoassay, which has a high sensitivity. false-positive results may occur in patients infected with blastomycosis and coccidioides species. tests for histoplasma antibodies by complement fixation or immunodiffusion may be negative in immunosuppressed, hiv-positive patients. the clinical presentation of coccidioidomycosis is variable. the chest radiograph may show focal pulmonary disease with focal alveolar infiltrates, adenopathy, and intrapulmonary cavities or, alternately, diffuse reticular infiltrates. diagnosis is made by isolation of the organism in sputum or bal fluid. disseminated disease is identified by isolating the fungus in blood, urine, or cerebrospinal fluid. serologic tests may also be used for diagnosis. blastomycosis presents in patients who have advanced hiv infection, when cd lymphocyte counts are usually less than cells/ml. clinical symptoms include cough, fever, dyspnea, and weight loss. patients may present late in respiratory failure. disseminated disease can occur with both pulmonary and extrapulmonary features. there is frequently multiple involvement of the skin, liver, brain, and meninges. chest radiographic abnormalities include focal pneumonic change, miliary shadowing, or diffuse interstitial infiltrates. diagnosis is made by culture from bal fluid, skin, and blood. in this infection, cytologic or histologic diagnosis is important for early diagnosis, because culture of the organism may take - weeks. the mortality rate is high in patients with disseminated infection. p. marneffei infection is particularly common in southeast asia. most hiv-infected patients present with disseminated infection and solitary skin or oral mucosal lesions, or with multiple infiltrates in the liver or spleen, or bone marrow (leading to presentation with pancytopenia). pulmonary infection has no specific clinical features, and chest radiographs may be negative or show diffuse, small nodular infiltrates. diagnosis is made by identifying the organism in bone marrow, skin biopsy samples, blood films, or bal fluid. the differential diagnosis of p. marneffei infection includes both pcp and tuberculosis. these occur with equal frequency in hiv-infected and non-hiv-infected patients; however, respiratory complications after influenza infection are increased in patients affected with underlying conditions such as cardiac or pulmonary disease and immunosuppression. in prospective studies of hivinfected patients undergoing bronchoscopy for evaluation of suspected lower respiratory tract disease, the communityacquired respiratory viral infections (i.e., influenza, parainfluenza, respiratory syncytial virus, rhinovirus, coronavirus and adenovirus) are found only rarely, if at all. cmv chronically infects most hiv-infected individuals, and up to % of homosexual hiv-infected men shed cmv intermittently in urine, semen, and saliva. clinical disease may be caused by cmv in patients who have advanced hiv infection and cd counts < cells/ml. chorioretinitis is most frequently encountered, but encephalitis, adrenalitis, esophagitis, and colitis are also seen. frequently, cmv is isolated from bal fluid, being found in % of samples from patients with cd counts < cells/ml. however, the role of cmv in causing disease in this context is unclear (see later). in patients who have cmv as the sole identified pathogen, clinical presentation and chest radiographic abnormalities (usually diffuse interstitial infiltrates) are nonspecific. diagnosis of cmv pneumonitis is made by identifying characteristic intranuclear and intracytoplasmic inclusions, not only in cells in bal fluid but also in lung biopsy specimens (figure - ). pulmonary involvement with leishmania species may rarely occur as part of the syndrome of visceral leishmaniasis in hiv-infected patients. patients usually have advanced hiv disease with cd lymphocyte counts less than cells/ml and present with unexplained fever, splenomegaly, and leukopenia. respiratory symptoms are often absent. diagnosis of visceral leishmaniasis is most often made by staining a splenic or bone marrow aspirate and subsequent culture. occasionally, the parasite is found by chance in a skin or rectal biopsy or bal fluid taken for other purposes. the chest radiograph may be negative or show reticular-nodular infiltrates. toxoplasma gondii infection in patients who have aids usually occurs as a result of reactivation of latent, intracellular protozoa acquired in a primary infection. patients are invariably systemically unwell, with malaise and pyrexia. clinical disease in association with hiv infection is most commonly seen in the central nervous system, where it produces single or multiple abscesses. multisystem infection with t. gondii is uncommon in patients who have hiv infection. toxoplasmic pneumonia is frequently difficult to distinguish from pcp. nonproductive cough and dyspnea are the symptoms most commonly reported. chest radiographic abnormalities include diffuse interstitial infiltrates indistinguishable from those of pcp ( figure - ) , as well as micronodular infiltrates, a coarse nodular infiltrate, cavitary change, and lobar consolidation. the diagnosis is made by hematoxylin-eosin or giemsa staining of bal fluid that reveals cysts and trophozoites of t. gondii. staining of bal fluid is not always positive; the diagnostic yield is increased either by staining of transbronchial biopsy material or by performing nucleic acid amplification procedures such as pcr to detect t. gondii dna in bal fluid. the most frequent manifestation of infection with cryptosporidium species in hiv infection is a noninflammatory diarrhea that may be of high volume, intractable, and life threatening. cryptosporidium species may colonize epithelial surfaces, including the trachea and lungs, occasionally resulting in pulmonary infection. most cases of pulmonary cryptosporidiosis have co-pathology such as pcp or bacterial pneumonia; ascertaining the exact role of cryptosporidiosis as the cause of respiratory symptoms may be difficult. diagnosis is made by ziehl-neelsen or auramine-rhodamine staining of bal fluid or transbronchial biopsy specimens. pulmonary microsporidia infection may occur as part of systemic dissemination from gastrointestinal infection with septata intestinalis or encephalitozoon hellem. the organism may be identified by conventional staining in bal fluid. electron microscopy is necessary to distinguish the two species. the nematode strong yloides stercoralis is endemic in warm countries worldwide. in immunosuppressed patients, the organism has an increased ability to reproduce parthenogenetically in the gastrointestinal tract without the need for repeated exposure to new infection-so-called autoinfection. this results in a great increase in worm load and a hyperinfective state ensues; massive acute dissemination with s. stercoralis may occur in the lungs, kidneys, pancreas, and brain. although infection with s. stercoralis is more severe in immunocompromised patients, it is no more common in patients who have hiv infection. presentation with hyperinfection may be with fever, hypotension secondary to bacterial sepsis, or disseminated intravascular coagulation. the clinical features of respiratory s. stercoralis infection are nonspecific. s. stercoralis in sputum or bal fluid (figure - ) may be identified in hiv-positive patients in the absence of symptoms elsewhere; this can predate disseminated infection and, as such, requires prompt treatment. it is apparent from the foregoing discussion that hiv-related pneumonia of any cause may present in a similar manner. a wide range of investigations is available to aid diagnosis. these are listed in table - . if the subject is producing sputum, it is important to obtain samples for bacterial and mycobacterial detection. in up to one third of cases, these will assist in diagnosis. three samples on consecutive days (preferably either with overnight or early morning production) is the critical first step in the diagnosis of pulmonary tuberculosis. this is considerably easier and safer for health care personnel than obtaining hypertonic saline-induced sputum or bal fluid. blood cultures are also important, because very high rates of bacteremia have been reported in both bacterial and mycobacterial disease (see earlier). a patient who is initially seen with symptoms and signs consistent with pneumonia should have chest radiography and arterial oxygen assessments performed at his or her first consultation. the question at this stage is usually whether this infectious episode is due to bacterial infection, tuberculosis, or pcp. in general, alveolar and interstitial shadowing is taken as evidence for pcp, although important caveats apply. transcutaneous pulse oximetry and arterial blood gas analysis are useful tests for hypoxemia. they can be used to distinguish an alveolar condition (i.e., pcp) from bacterial pneumonia. the alveolitis produces a greater impairment of oxygen transfer (especially during exercise), such that for a given clinical situation there will be more hypoxemia and a wider alveolararterial oxygen gradient (a-ao ) in those with pcp. with pulse oximetry, this manifests as low oxygen saturations at rest that decrease further with exercise. in general, more information can be obtained from arterial blood gas analysis, although this advantage is offset by the need for direct arterial puncture. of patients with pcp, fewer than % have a normal pao and a normal a-ao . these measures are sensitive, although not particularly specific for pcp, and similar results may occur with bacterial pneumonia, pulmonary kaposi sarcoma, and m. tuberculosis infection. the diagnostic value of identifying exercise-induced desaturation, measured by transcutaneous oximetry, has been validated only in hiv-infected patients who have pcp and a normal or "near normal" appearance on chest radiographs. the test's value has not been confirmed in patients with abnormal chest radiographs because of pcp or other pathogens. exercise-induced desaturation may persist for many weeks after treatment and recovery from pcp, even in the absence of active pulmonary disease. abnormalities of lung function are well documented with hiv infection. the most common of these relate to tests measuring gas exchange, rather than the size of the conducting airways. in general, an overall reduction in diffusing capacity for carbon monoxide (dlco) occurs at all stages of hiv infection, with the largest changes found in hiv-infected patients with pcp. thus, to some extent, patients who have probable pcp can be differentiated and treatment guided. a normal dlco in an individual who has symptoms but a negative or unchanged chest radiograph makes the diagnosis of pcp extremely unlikely. data from the north american pspc cohort study suggest that individuals with rapid rates of decline in dlco are at an increased risk for development of pcp. recent work suggests that hiv-infected smokers are at increased risk of early-onset emphysema. smoking history must, therefore, be taken into account when assessing a patient's lung function results in the context of possible pcp. high-resolution (fine-cut) ct scanning of the chest may be helpful when the chest radiograph is normal, unchanged, or equivocal. the characteristic appearance of an alveolitis (i.e., areas of ground-glass attenuation through which the pulmonary vessels can be clearly identified) may be present, which indicates active pulmonary disease (figure - ). this feature, however, is neither sensitive nor specific for pcp, although its sensitivity can be improved if evidence for reticulation and/or small cystic lesions is added to this. hence, a negative test result implies an alternative diagnosis. in the context of an hiv-infected patient who is seen with an acute or subacute pneumonitis, an elevated serum lactate dehydrogenase (ldh) enzyme level is strongly suggestive of pcp. when interpreting such results, it is important to remember that other pulmonary disease processes (e.g., pulmonary embolism, nonspecific pneumonitis, and bacterial and mycobacterial pneumonia) and extrapulmonary disease (castleman disease and lymphoma) may also cause elevations of ldh and may need to be considered in the correct clinical context. from the previous information, it is evident that noninvasive tests cannot reliably distinguish the different infecting agents from each other but may be useful in excluding acute opportunistic disease. thus, the clinician is left with either proceeding to diagnostic lung fluid or tissue sampling (by either induced sputum collection or bronchoscopy and bal with or without transbronchial biopsy table - ) or treating an unknown condition empirically. haart has also altered the investigation of respiratory disease. the numbers of invasive procedures performed are falling and tend to be in patients spontaneously expectorated sputum is inadequate for diagnosis of pcp. sputum induction by inhalation of ultrasonically nebulized hypertonic saline may provide a suitable specimen (see table - ). the technique requires close attention to detail and is much less useful when samples are purulent. sputum induction must be carried out away from other immunosuppressed patients and health care workers, ideally in a room with separate negative-pressure ventilation, to reduce the risk of nosocomial transmission of tuberculosis. although very specific (> %), the sensitivity of induced sputum varies widely ( - %), and therefore a negative result for p. jirovecii prompts further diagnostic studies. the use of immunofluorescence staining enhances the yield of induced sputum compared with standard cytochemistry. fiberoptic bronchoscopy with bal is commonly used to diagnose hiv-related pulmonary disease. when a good "wedged" sample is obtained, the test has a sensitivity of more than % for detection of p. jirovecii (figure - ). just as with induced sputum, fluorescent staining methods increase the diagnostic yield, which makes it the procedure of choice in most centers. more technically demanding (both of the patient and the operator) than induced sputum collection, bronchoscopy and bal have the advantage that direct inspection of the upper airway and bronchial tree can be performed and, if necessary, biopsies taken. transbronchial biopsies may marginally increase the diagnostic yield of the procedure. this is relevant for the diagnosis of mycobacterial disease, although the relatively high complication rate in hiv-infected individuals (pneumothorax and the possibility of significant pulmonary hemorrhage in up to %) outweighs the advantages of the technique for routine purposes. samples of bal fluid are examined for bacteria, mycobacteria, viruses, fungi, and protozoa. inspection of the cellular component may also provide etiologic clues-cooperation of a pathology department with experience in opportunistic infection diagnosis is vital. the drug interactions associated with antiretroviral protease inhibitor (pi) therapy mean that special care should be exercised when sedation is used with either benzodiazepine or opiate drugs. prolonged sedation and life-threatening arrhythmias have been reported. a diagnostic strategy, therefore, includes sputum induction and, if results are nondiagnostic or if the test is unavailable, bronchoscopy and bal. if this does not yield a result, consideration is given to either a repeat bronchoscopy and bal with transbronchial biopsies or surgical biopsy. the latter can be performed as either an open lung or thoracoscopic procedure. surgical biopsy has a high sensitivity. although empirical therapy is usually reserved for the management of presumed bacterial pneumonias, and at first sight may seem unwise when dealing with possible opportunistic infection, in reality pcp is almost invariably a diagnosis of exclusion, and certain clinical and laboratory features may guide the assessment of an hiv-infected individual's risk for this condition. the likelihood that p. jirovecii is the causative organism increases if the subject is not taking effective anti-pneumocystis drug prophylaxis or has a previous medical history with clinical or laboratory features that suggest systemic immunosuppression (i.e., recurrent oral thrush, longstanding fever of unknown cause, clinical aids, or blood cd count < cells/ml). hence, some centers advocate use of empirical therapy for hiv-infected patients who are seen with symptoms and chest radiographic and blood gas abnormalities typical of mild pcp, without the need for bronchoscopy. invasive measures are reserved for those with an atypical radiographic presentation, those who fail to respond to empirical therapy by day , and those who deteriorate at any stage. most clinicians in north america and the united kingdom would seek to obtain a confirmed diagnosis in every case of suspected pcp. in practice, both strategies seem to be equally effective, although a number of caveats should be borne in mind when empirical treatment is given for pcp. patients who have pcp typically take - days to show clinical signs of improvement, so a bronchoscopically proven diagnosis ensures that the treatment being given is correct, particularly in the first few days of therapy, when it may not be well tolerated. in addition, the diagnosis of pcp has implications for the infected individual, because it may influence the decision to start either haart or anti-pneumocystis prophylaxis. finally, empirical therapy requires the patient to be maximally adherent to treatment, because nonresolution of symptoms may be seen as failure of therapy rather than of compliance. molecular biologic techniques (such as pcr) are increasingly used in the diagnosis of respiratory disease. two examples of this are dna amplification of loci of the p. jirovecii and m. tuberculosis genomes. the advantages of molecular methods are that the diagnosis may be made by use of samples that are more readily obtained than bal fluid (i.e., expectorated sputum or nasopharyngeal secretions) and also that these methods are rapid (the answer may be available within a working day, compared with conventional mycobacterial culture, which may take weeks). despite encouraging results in the research setting (sensitivity and specificity have been reported as - % and - %, respectively), problems persist when these techniques are applied to routine diagnostic samples. these include extraction of nucleic acid from clinical material, cross-contamination with the products of previous assays, and clinical interpretation of a test result. currently, treatment individuals infected with hiv, compared with the non-hivinfected general population, have an increased likelihood of adverse reactions to therapy. this includes tmp/smx (see later) and other antibacterial and antimycobacterial agents. in addition, there are complex drug interactions with other medications, particularly components of haart. before instituting therapy for any infectious complication in an hivinfected individual, it is important to consult with a physician experienced in the care of patients with hiv infection and to seek advice from a specialist pharmacist. the main organisms causing pneumonia in hiv-infected individuals are similar to those found in the general population with community-acquired pneumonia. thus, bacterial pneumonia in hiv-infected patients should be treated in a similar manner to that in hiv-negative individuals, by use of the published american thoracic society (ats) and british thoracic society (bts) guidelines. in addition, expert advice on local antibiotic resistance patterns should be sought from infectious disease or microbiology colleagues, because treatment is usually begun on an empirical basis before the causative organism is identified and antibiotic sensitivities known. the same clinical and laboratory prognostic indices that are described for the general population apply to hiv-infected patients and should be documented on presentation. response to appropriate antibiotic therapy is usually rapid and is similar to that seen in the non-hiv-infected individual. early relapse of infection after successful treatment is well described. those hiv-infected patients who have presumed pcp and are being treated empirically with high-dose tmp/ smx, and who have infection with either s. pneumoniae or h. influenzae rather than p. jirovecii, may also improve. in addition, in those patients who are treated with benzylpenicillin for proven s. pneumoniae pneumonia but do not respond, and penicillin resistance can be discounted as the cause, it is important to consider whether there is a second pathologic process, such as pcp. co-pathogens are reported in up to % of cases of pneumonia. before instituting treatment, assessment of the severity of pcp should be performed on the basis of history, findings on examination, arterial blood gas estimations, and chest radiographic abnormalities. patients can then be stratified into those with mild, moderate, or severe disease (table - ) . this is important, because some drugs are of unproven benefit and others are known to be ineffective for the treatment of severe disease. in addition, adjuvant glucocorticoid therapy may be given to patients with moderate or severe pneumonia. patients with glucose- -phosphate dehydrogenase deficiency should not receive tmp/smx, dapsone, or primaquine, because these drugs increase the risk of hemolysis. several drugs are effective in the treatment of pcp. tmp/ smx is the drug of first choice (tables - and - ). overall it is effective in - % of individuals when used as firstline therapy. adverse reactions to tmp/smx are common and usually become apparent between days and of treatment. neutropenia and anemia (in up to % of patients), rash and fever (up to %), and biochemical abnormalities of liver function (up to %) are the most frequent adverse reactions. hematologic toxicity induced by tmp/smx is neither attenuated nor prevented by coadministration of folic or folinic acid. furthermore, the use of these agents may be associated with reduced therapeutic success. during treatment with tmp/ smx, full blood count, liver function, and urea and electrolytes should be monitored at least twice weekly. it is not known why hiv-infected individuals, especially those with higher cd counts, have such a high frequency of adverse reactions to tmp/smx. the optimum strategy for an hiv-infected patient who has pcp and who becomes intolerant of high-dose tmp/smx has not been established. many physicians "treat through" minor rash, often adding an antihistamine and a short course of oral prednisolone ( mg every h, reducing to zero over days). if treatment with tmp/smx fails, or is not tolerated by the patient, several alternative therapies are available (see tables - and - ). the combination of clindamycin and primaquine is widely used for treatment of pcp whatever the severity, although there is no license in the united kingdom or united states for this indication. the combination is as effective as oral tmp/smx and oral trimethoprim-dapsone for the treatment of mild and moderate-severity disease. as a second-line treatment it is effective in up to % of patients. methemoglobinemia caused by primaquine occurs in up to % of patients. if mg four times daily of primaquine is used, rather than mg four times daily, the likelihood of methemoglobinemia is reduced. diarrhea develops in up to % of patients receiving clindamycin. if this occurs, stool samples should be analyzed for the presence of clostridium difficile toxin. this oral combination is as effective as oral tmp/smx and oral clindamycin plus primaquine (see earlier) for treatment of mild and moderate-severity pcp. the combination has not been shown to be effective in patients who have severe pcp. most patients experience methemoglobinemia (caused by dapsone), which is usually asymptomatic. up to one half of patients have mild hyperkalemia (< . mmol/l) caused by trimethoprim. a methotrexate analog, trimetrexate, is given intravenously together with folinic acid "rescue" to protect human cells from trimetrexate-induced toxicity. in patients who have moderate regimen recommended by cdc/ nih/idsa, widely used in usa methylprednisolone iv at % of dose given above for prednisolone methylprednisolone prednisolone iv q h, days - . iv days - then g po q h reducing to , days - nb, none of these regimens for adjuvant glucocorticoid therapy have been compared in prospective clinical trials. to severe disease, trimetrexate-folinic acid is less effective than high-dose tmp/smx, but serious treatment-limiting hematologic toxicity occurs less frequently with trimetrexate-folinic acid. atovaquone is licensed for the treatment of mild and moderate-severity pcp in patients who are intolerant of tmp/ smx. in tablet formulation (no longer available), this drug was less effective but was better tolerated than tmp/smx or intravenous pentamidine for treatment of mild or moderateseverity pcp (see tables - and - ). there are no data from prospective studies that compare the liquid formulation (which has better bioavailability) with other treatment regimens. common adverse reactions include rash, fever, nausea and vomiting, and constipation. absorption of atovaquone is increased if it is taken with food. intravenous pentamidine is now seldom used for the treatment of mild or moderate-severity pcp because of its toxicity. intravenous pentamidine may be used in patients who have severe pcp, despite its toxicity, if other agents have failed (see tables - and - ). nephrotoxicity develops in almost % of patients given intravenous pentamidine (indicated by elevation in serum creatinine), leukopenia develops in approximately half, and up to % have symptomatic hypotension or nausea and vomiting. hypoglycemia occurs in approximately % of patients. given the long half-life of the drug, this may occur up to several days after the discontinuation of treatment. pancreatitis is also a recognized side effect. for patients who have moderate and severe pcp, adjuvant glucocorticoid therapy reduces the risk of respiratory failure by up to half and the risk of death by up to one third (see tables - and - ). glucocorticoids are given to hivinfected patients with confirmed or suspected pcp who have a pao < . kpa (< mmhg) or an a-ao of > . kpa (< mmhg). oral or intravenous adjunctive therapy is given at the same time as (or within h of starting) specific anti-p. jirovecii therapy. clearly, in some patients treatment is commenced on a presumptive basis, pending confirmation of the diagnosis. in prospective studies, adjuvant glucocorticoids have not been shown to be of benefit in patients with mild pcp. however, it would be difficult to demonstrate this, given that survival in such cases approaches % with standard treatment. patients with mild pcp may be treated with oral tmp/smx as outpatients if they are able to manage at home, willing to attend the outpatient clinic for regular review, and that there is clinical and radiographic evidence of recovery. if the patient is intolerant of oral tmp/smx despite clinical recovery, either the treatment is given intravenously or treatment may be changed to oral clindamycin plus primaquine. all patients with moderate and severe pcp should be hospitalized and given intravenous tmp/smx or intravenous clindamycin and oral primaquine (plus adjuvant steroids). patients with moderate or severe disease who show clinical and radiographic response by day - of therapy may be switched to oral tmp/smx to complete the remaining days of treatment. if the patient has failed to respond within - days or deteriorates before this time while receiving tmp/smx, then treatment should be changed to clindamycin and primaquine or trimetrexate plus folinic acid. deterioration in a patient who is receiving anti-p. jirovecii therapy may occur for several reasons (table - ) . before ascribing deterioration to treatment failure and considering a change in therapy, these alternatives should be evaluated carefully. it is also important to consider treating any co-pathogens present in bal fluid, to perform bronchoscopy if the diagnosis was made empirically, to repeat the procedure, or to carry out open lung biopsy to confirm that the diagnosis is correct. most centers advocate admission to the icu for pcp with respiratory failure and for acute severe deterioration after bronchoscopy. the prognosis for severe pcp in such circumstances has improved over the past decade. this is likely because of a greater understanding of successful general icu management of respiratory failure and acute respiratory distress syndrome (ards) rather than specific improvements in pcp care. factors associated with poor outcome include increasing patient age, need for mechanical ventilation, and development of a pneumothorax. the latter reflects both the association between this complication and pcp, as well as the subsequent difficulty in successful mechanical ventilation of such individuals. the treatment of hiv-related mycobacterial disease is complex. not only do individuals have to take prolonged courses of relatively toxic agents, but also these antimycobacterial drugs have side effects similar to those of other prescribed in the developed world, isoniazid-related peripheral neuropathy is rare in hiv-negative subjects taking pyridoxine. the nucleoside reverse transcriptase inhibitors (rti) didanosine and stavudine, which are now less frequently used in the united states and the united kingdom but which remain a mainstay of haart in the developing world, can also cause a painful peripheral neuropathy. this complication develops in up to % of patients if stavudine and isoniazid are coadministered. rash, fever, and biochemical hepatitis are common adverse events with rifamycins, pyrazinamide, and isoniazid (occurring more frequently in patients with tuberculosis who have hiv infection with hepatitis c coinfection). the nonnucleoside rti drugs (e.g., nevirapine) have a similar toxicity profile. if treatment for both hiv and tuberculosis is co-administered, ascribing a cause may be problematic. drug-drug interactions between medications used to treat tuberculosis and hiv infection occur because of their common pathway of metabolism through the hepatic cytochrome p- enzyme system. rifampin is a potent inducer of this enzyme (rifabutin less so), which may result in subtherapeutic levels of nonnucleoside rti and pi antiretroviral drugs, with the potential for inadequate suppression of hiv replication and the development of resistance to hiv. in addition, the pi class of antiretroviral drugs inhibits the metabolism of rifamycins, which leads to increases in their plasma concentration and is associated with increased drug toxicity. the nonnucleoside rti drugs are inducers of this enzyme pathway. coadministration of rifabutin with efavirenz requires an increase in the dose of rifabutin to compensate for the increase in its metabolism induced by efavirenz (see later). the optimal duration of treatment of tuberculosis, by use of a rifamycin-based regimen, in a patient who has hiv infection is unknown. current recommendations (joint tuberculosis committee of the bts and the ats/cdc/infectious disease society of north america [idsa]) are to treat tuberculosis in hiv-infected patients in the same way as for the general population (i.e., for months for drug-sensitive pulmonary tb). in addition, ats/cdc/idsa guidelines recommend that treatment be extended to months in those who have cavitation on the original radiograph, continuing signs, or a positive culture after months of therapy. recent work has highlighted the increased risk for development of rifampin monoresistance in hiv-infected individuals on treatment. this is especially so if intermittent regimens are used and may arise from a lack of efficacy of the other drugs present in the combination (e.g., intermittent isoniazid). hence, daily medication regimens are recommended and should be closely supervised in all hiv-positive patients. it should be remembered that although rifabutin is usually given three times a week with ritonavir-boosted protease inhibitors, this seems to achieve adequate rifamycin levels; there have been no reports of this leading to rifamycin resistance in patients who are appropriately adherent. directly observed therapy (dot) is an important, although fairly labor-intensive, strategy that has the support of the world health organization. the best time to start therapy in patients being treated for tuberculosis is unknown. decision analyses show that early treatment with antiretroviral therapy leads to a marked reduction in further opportunistic disease. against this is balanced the risk of needing to discontinue antituberculosis therapy or hiv therapy because of drug toxicity or drug-drug interactions. iris is reported to be more likely if the treatments are started at the same time as each other. pragmatically, delaying the start of antiretroviral therapy simplifies patient management and may reduce or prevent adverse drug reactions and drug-drug interactions and may also reduce the risk of iris. on the basis of current evidence, patients with cd counts > cells/ml have a low risk of hiv disease progression or death during months of treatment for tuberculosis. in these patients, the cd count should be closely monitored, and antiretroviral therapy may be deferred until treatment for tuberculosis is completed. in patients who have cd counts from - cells/ml, many centers currently delay starting antiretroviral therapy until after the first months of treatment for tuberculosis have been completed; patients are given concomitant pcp prophylaxis. in patients who have cd counts of < cells/ml, antiretroviral therapy is started as soon as possible after beginning treatment for tuberculosis. this is based on evidence that shows a significant short-term risk of hiv disease progression and death in this patient group if antiretroviral therapy is delayed. two options exist for starting antiretroviral therapy in a patient already being treated for tuberculosis. first, the rifampin-based regimen is continued, and antiretroviral therapy is commenced, for example, with a combination of two nucleoside rtis and a non-nucleoside rti, such as efavirenz (if the patient weighs < kg, the efavirenz dose is often increased to mg once daily to compensate for rifampin-induced metabolism of efavirenz). alternately, the rifampin is stopped and rifabutin is started: antiretroviral therapy is given, with a combination of two nucleoside rti drugs and either a single ritonavir-boosted pi or a nonnucleoside rti. here the dose of rifabutin is adjusted to take into account the pharmacokinetic effect of the co-administered drug. with a boosted pi, it is usually prescribed at a dose of mg three times weekly and with efavirenz it is increased to mg once a day. before the advent of antiretroviral therapy, it was recognized by tuberculosis physicians that patients who were apparently responding to their antimycobacterial treatment would sometimes have a short period of clinical deterioration develop. this "paradoxical reaction" (in the face of overall treatment response) was seen as an interesting and probable immunebased phenomenon of generally little consequence. the widespread introduction of haart has led to an increased awareness by clinicians of similar, but generally more severe, events in hiv-infected individuals. in the context of hiv, these are termed iris, or immune reconstitution disease. they can present in a number of ways and with a range of opportunistic conditions. perhaps the most common of these is similar to a paradoxical reaction. here, after initiation of antiretroviral therapy in a patient being treated for tuberculosis, for example, there arises the return of the original or the development of new symptoms and signs. these are often of a systemic nature and may be associated with marked radiographic changes. examples of this include fever, dyspnea, lymphadenopathy, effusions, parenchymal pulmonary infiltrates, or expansion of cerebral tuberculomas. this form of iris is seen most frequently with mycobacteria (commonly tuberculosis or mac), fungi (notably, cryptococcus), and viruses (hepatitis and herpes viridae). iris develops in up to one third of hiv-infected patients being treated for tuberculosis when antiretroviral therapy is started. the median onset of tuberculosis-related iris is approximately weeks from beginning antituberculosis treatment or weeks from commencing haart. it seems to be more likely in patients who have disseminated tuberculosis (and hence presumably more antigen present as well as more potential for significant inflammatory reactions) and a lower baseline blood cd count. a rapid fall in hiv load, as well as a large increase in cd counts in response to haart, may also predict iris. the relationship between early use of haart and low blood cd counts suggests that care must be taken when starting antiretrovirals in patients with tb at sites where rapid expansion of an inflammatory mass could be life threatening. examples of this would include cerebral, pericardial, or peritracheal disease (figure - ) . it is important to note that iris is currently a diagnosis of exclusion. there is no laboratory test available to assist with this; it should be made only after progressive or (multi) drug-resistant tuberculosis, poor drug adherence (to either antituberculosis or antiretroviral agents) and drug absorption, or an alternative pathologic process have been excluded as an explanation for the presentation. criteria have been drawn up that seek to provide clinical diagnostic criteria (table - ) . the mechanism leading to iris is unclear. it is not due to failure of treatment of tuberculosis or to another disease process; if anything, it is most likely to represent an exuberant and uncontrolled response to mycobacterial antigens (from both dead and live organisms). current treatments include nonsteroidal antiinflammatory drugs or glucocorticoids. the latter are undoubtedly effective, although they can lead to hyperglycemia and hypertension. recent preliminary data suggest that the leukotriene receptor antagonist montelukast may be of benefit in iris (this drug is unlicensed for this indication). recurrent aspiration of lymph nodes or effusion may also be needed. although iris is often self-limiting, it may persist for several months. rarely, temporary discontinuation of antiretroviral therapy is required. in this situation there may be precipitous falls in cd counts; patients are at risk of other opportunistic infections. attention has also focused on what is possibly more of a concern-the form of iris referred to as "unmasking phenomenon." here, individuals with presumably latent tuberculosis infection who start haart have systemic active (and often infectious) tuberculosis develop within a -month period. although it is likely that the patient's disease would have presented in time anyway and that some of the reported cases may, in fact, represent ascertainment bias, the current view is that this is real and represents an adverse effect of haart. given that the people most at risk live in countries with limited facilities for pre-haart screening, this has major implications for antiretroviral therapy roll-out programs in resource-poor areas. combination antimycobacterial therapy by itself does not cure mac infection. a commonly used regimen is oral rifabutin, mg once daily, with oral ethambutol, mg/kg once daily, and oral clarithromycin, mg once daily or every h. if clarithromycin is not used, oral rifabutin, mg once daily, is given-the lower dose adjusting for yet more drug-drug interactions. use of three drugs has no impact on overall outcome, although it reduces the risk of resistance and possibly enhances early mycobacterial killing. in patients severely compromised by symptoms, intravenous amikacin, . mg/kg once daily for - weeks, is also given. trough blood levels must be measured to ensure toxic accumulation of amikacin does not occur. fluoroquinolones such as moxifloxacin or levofloxacin may be extremely useful, because they have good antimycobacterial activity with limited side effects. at present, many of these agents are not licensed for this indication. given the concerns over xdr tuberculosis, it is important to ensure that patients are adherent to such treatments, and hence reduce the risk of fluoroquinolone resistance developing. a frequently used regimen includes rifampin, isoniazid, and ethambutol in conventional doses; all drugs are given by mouth. the treatment regimens for fungal infections complicating hiv infection are shown in table - . after initial treatment of cryptococcal infection, there is a high likelihood of relapse of infection; hence, lifelong secondary preventative therapy is needed unless antiretroviral therapy is commenced and results in sustained improvements in cd counts (> cells/ml) and suppression of hiv load in peripheral blood. secondary prophylaxis is most often oral fluconazole - mg four times daily. just as with mycobacterial disease, "late" iris events can occur after months or even years. these should be investigated to exclude active disease and other conditions. oral itraconazole, mg twice daily, is the current treatment of choice. the dose is adjusted to achieve blood trough drug levels that are above the standard lowest effective concentration. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. treatment of this infection is difficult. after initial treatment with amphotericin b, itraconazole or fluconazole may be given for long-term suppression. the overall prognosis is poor, with a % mortality rate despite therapy. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. oral itraconazole has now replaced amphotericin b as the treatment of choice for p. marneffei infection, apart from the subgroup who are acutely unwell. fluconazole is less effective than itraconazole. after initial treatment, lifelong suppressive therapy with itraconazole is needed. there are no data on the impact of antiretroviral therapy on which to base decisions about discontinuation of secondary prophylaxis. the treatment regimens are shown in table - . a combination of sulfadiazine and pyrimethamine is the regimen of choice for t. gondii infection. the most frequent dose-limiting side effects are rash and fever. adequate hydration must be maintained to avoid the risk of sulfadiazine crystalluria and obstructive uropathy. alternate regimens are given in table - . once treatment is completed, lifelong maintenance is necessary to prevent relapse, unless antiretroviral therapy achieves adequate immune restoration (blood cd count > cells/ml and undetectable hiv load). visceral leishmaniasis is usually treated with liposomal amphotericin b, although this is still associated with a high rate of relapse. second-line therapy (or first-line in resourcepoor environments) is to use sodium stibogluconate (see table - ) . phenomena temporally associated with starting haart. this includes, but is not limited to: . new or enlarging lymphadenopathy, cold abscesses, or other focal tissue involvement . new or worsening central nervous system disease . new or worsening radiological features of tuberculosis . new or worsening serositis (pleural effusion, ascites, pericardial effusion, or arthritis. . new or worsening constitutional symptoms such as fever, night sweats, and/or weight loss . retrospective review indicating that a clinical or radiologic deterioration occurred with no change having been made to tuberculosis treatment c. immune restoration, e.g., a rise in cd lymphocyte count in response to haart d. a fall in hiv "viral load" in response to haart alternative diagnoses to be excluded progressive underlying infection treatment failure due to drug resistance (mdr or xdr) treatment failure from poor adherence adverse drug reaction another diagnosis coexisting (e.g., non-hodgkin lymphoma) the treatment of choice is ivermectin. risk of treatment failure with thiabendazole in hiv-infected individuals is higher than that in non-hiv-infected patients. cytomegalovirus pneumonitis is treated with intravenous ganciclovir, mg/kg every h, for days. drug-induced neutropenia is managed with granulocyte colony-stimulating factor. some centers use valganciclovir, an oral formulation of ganciclovir, at a dose of mg orally every h, to treat cmv pneumonitis. side effects and their management are as for ganciclovir. there are no data that demonstrate efficacy for cidofovir for treatment of cmv pneumonitis, but this agent is used as second-line therapy in many centers. phosphonoformate (foscarnet) can be used for treatment of cmv endorgan disease (e.g., pneumonitis), although it has an extensive toxicity profile. it is also a moderately effective antiretroviral agent. this effect is occasionally used as an adjunct in controlling nonresponsive viral infections. within the past few years, drug therapy has radically altered the depressingly predictable nature of progressive hiv infection. combinations of specific opportunistic infection prophylaxis and antiretroviral therapy can reduce both the incidence and the mortality associated with common conditions. the observational north american macs cohort demonstrated that the risk of pcp in individuals with blood cd counts of < cells/ml can be reduced almost fourfold if both specific prophylaxis and haart are taken (from % to %). however, as common conditions are prevented, so other less treatable illnesses may arise. the initial impact of p. jirovecii prophylaxis was a reduction in the incidence of pcp at the expense of an increase in cases of disseminated mac infection, cmv infection, esophageal candidiasis, and wasting syndrome. new prophylactic therapies targeting those conditions associated with high morbidity and mortality (in particular mac) have further improved survival. it has become apparent that specific infection prophylaxis may also confer protection against other agents. this "cross- prophylaxis" is particularly seen with the use of tmp/smx for pneumocystis, which also provides cover against cerebral toxoplasmosis and several common bacterial infections (although not s. pneumoniae) and with macrolides for mac infection, which further reduce the incidence of bacterial disease and also pcp. use of large amounts of antibiotic raises the possibility of future widespread drug resistance. this is clearly of concern, and recent reports suggest that, indeed, in some parts of the world the incidence of pneumococcal tmp/smx resistance is rising. current preventive therapies pertinent to lung disease focus on p. jirovecii, mac, m. tuberculosis, and certain bacteria (table - ). numerous studies have demonstrated the greatly increased risk in subjects who do not take adequate drug therapy with blood cd counts < cells/ml. clinical symptoms are also an independent risk factor for pcp, and hence the current guidelines recommend lifelong prophylaxis against p. jirovecii in hiv-infected adults who have had prior pcp, cd counts < cells/ml, constitutional symptoms (documented oral thrush or fever of unknown cause of < . c that persists for more than weeks), or clinical aids. the importance of secondary prophylaxis (i.e., used after an episode of pcp) becomes clear from historical data, which indicate a % risk of relapse in the first months after infection. the increase in systemic and local immunity that occurs with haart has led to several studies evaluating the need for prolonged prophylaxis in individuals with sustained elevations in blood cd counts and low hiv rna load. in summary, it seems that both primary and secondary pcp prophylaxis can be discontinued once cd counts are > cells/ml for more than months. a caveat to this is that the patient should have a low or undetectable hiv rna load, that the cd percentage is stable or rising and is > %, and that the individual plans to continue haart long term with good adherence. the risk of pcp recurrence is real if the cd count falls below cells/ml. if this does happen, pcp prophylaxis should be restarted. similar algorithms have been successfully used for all the major infections except tuberculosis. they all rely on an estimation of the general blood cd count above which clinical disease is highly unlikely. for example, secondary prophylaxis of mac may be discontinued once the blood cd count is consistently > cells/ml. this is a general guideline, however, and patients must be assessed on an individual basis. as with treatment strategies, tmp/smx is the drug of choice for prophylaxis (table - ) . it has the advantages of being highly effective for both primary and secondary prophylaxis (with -year risk of pcp while on the drug being . and . %, respectively). it is cheap, can be taken orally, acts systemically, and provides some cross-prophylaxis against other infections, such as toxoplasmosis, salmonella species, staphylococcus species, and h. influenzae. its main disadvantage is that adverse reactions are common (see earlier), occurring in up to % of individuals taking the prophylactic dose. the standard dose of tmp/smx is one double-strength tablet ( mg trimethoprim, mg sulfamethoxazole) per day. other regimens have been tried; these include one "double-strength" tablet three times weekly and one single-strength tablet per day. in general, when used for primary prophylaxis, these regimens are tolerated well (if not better than the standard) and seem as efficacious as one double-strength tablet per day. the data are less clear on secondary prophylaxis, in which subjects are at a much higher risk of recurrent pcp. attempts to desensitize patients who are intolerant of tmp/ smx have met with some success. in patients who cannot tolerate tmp/smx, dapsone is a safe and inexpensive alternative. it has been studied in a number of trials as both primary and secondary prophylaxis and is effective at an oral dose of mg/day. when combined with pyrimethamine ( mg three times weekly), it provides a degree of cross-prophylaxis against toxoplasmosis. before starting dapsone, patients are tested for glucose- -phosphate dehydrogenase deficiency. nebulized pentamidine has largely fallen from use as a prophylactic agent. this is despite it being better tolerated and having a similar efficacy to tmp/smx for primary preventive therapy. however, its breakthrough rate is higher in subjects who have lower cd counts (i.e., < cells/ml) and in those who take it as secondary prophylaxis. other disadvantages include equipment costs and complexity (alveolar deposition is crucial, and hence the nebulizer system used is important), the risk of transmission of respiratory disease (e.g., tuberculosis) to other patients and staff during the nebulization procedure, an alteration in the clinical presentation of pcp while on pentamidine (increased frequency of radiographic upper zone shadowing, increased incidence of pneumothorax), and a lack of systemic protection against pneumocystis and other infectious agents. there is also an acute bronchoconstriction effect during nebulization. long-term follow-up studies have not demonstrated any significant negative effect on lung function. atovaquone oral suspension is used as a second-line prophylactic agent in subjects intolerant of tmp/smx. it seems to have similar efficacy to dapsone (given together with weekly pyrimethamine), with a reduced incidence of side effects, of which the most frequent are rash, fever, and gastrointestinal disturbance. azithromycin is used in many centers as a third-line prophylactic agent. it is given at a dose of mg once weekly, and may provide protection against some bacterial infections, as well as mac. a low blood cd count (< cells/ml) is the current best laboratory predictor of prophylaxis failure. this is not particularly surprising given that the median blood cd count of subjects not on prophylaxis who have pcp develop is below cells/ml. persistent fever of unknown cause is an important clinical risk factor for pcp. used as preventive therapy, tmp/ smx significantly reduces the chance for development of pneumocystis. it is, therefore, vital that subjects who are most vulnerable be encouraged to use this drug on a regular basis. the pspc cohort study revealed that % of subjects with a cd count < cells/ml were not receiving any form of pcp prophylaxis. the effective and safe (i.e., replication incompetent) bacterial vaccines that are available would be expected to be widely used to prevent hiv-related disease. in fact, uptake of both pneumococcal and the h. influenzae type b (hib) vaccines is poor (current estimates for the former are at most only % of the infected population with the recommended -valent vaccine). one reason for this may be that the protection conferred by vaccination ( %) in the general population is not seen in immunosuppressed hiv-infected individuals, reflecting their inability to generate adequate memory b-cell responses (especially those subjects with cd counts < cells/ml). however, in north america, cdc/idsa recommend the pneumococcal vaccine as a single dose as soon as hiv infection is diagnosed, with a booster at years, or if an individual's blood cd count was < cells/ml and subsequently increased on haart. several studies show pneumococcal immunization reduces the risk of invasive pneumococcal infection in this population. this does not seem to be the case in a developing world setting, where not only is the -valent vaccine ineffective against both invasive and noninvasive pneumococcal disease, but the overall incidence of pneumonia is increased. infection with h. influenzae type b is much less common in hiv-infected adults and, therefore, immunization with hib vaccine is not routinely recommended. there is little evidence to suggest that the high frequency of bacterial infections in the hiv population is related to bacterial colonization. therefore, continuous antibiotics are rarely indicated, although both tmp/smx and the macrolides (clarithromycin and azithromycin) given as long-term prophylaxis for opportunistic infections have been shown to reduce the incidence of bacterial pneumonia, sinusitis/otitis media, and infectious diarrhea. the use of tmp/smx also confers a survival advantage in many studies performed in resource-poor settings. there is little evidence, however, that tmp/smx protects against pneumococcal infection. the interaction between hiv and tuberculosis is of fundamental importance, because the annual risk for the development of clinical tuberculosis in a given individual is estimated to be - % (i.e., similar to a non-hiv-infected subject's lifetime risk). hiv-infected individuals with pulmonary tuberculosis are less likely to be smear positive than their hiv-negative counterparts, although they can still transmit tuberculosis. thus, within a community, tuberculosis prevention involves case finding and treatment of active disease, as well as specific prophylactic drug therapies for those exposed. if possible, hiv-positive subjects should make every effort to avoid encountering tuberculosis (e.g., at work, homeless shelter, health care facility). one of the problems with standard methods of tuberculosis contact tracing in hiv infection is that both tuberculin skin test results and chest radiology may be unreliable. however, in the absence of bacillus calmette-guérin (bcg) immunization, a positive ppd (e.g., < mm induration with tuberculin units) indicates a greatly increased risk ( -to -fold compared with nonanergic, ppd-negative, hiv-infected subjects) of future active disease. the chance that hiv-infected subjects may contract disseminated infection if given bcg means that (having excluded active infection) the only option in these circumstances is to use a preventative drug regimen. options include at least months of isoniazid (together with pyridoxine to prevent peripheral neuropathy). this is safe and well tolerated, although compliance is a problem (especially with regimens longer than months), and dot may need to be instituted (e.g., mg isoniazid twice weekly). there is little evidence to suggest that this single-agent regimen leads to isoniazid resistance, which probably reflects the low mycobacterial load present in such individuals. attempts to shorten the length of treatment for latent infection have produced variable results. recent studies used rifampin and pyrazinamide for short-course prophylaxis ( months). this was as effective as months of isoniazid in hiv-coinfected individuals, although it was associated with fatal hepatotoxicity (almost exclusively in the hiv-negative population). hence, it is currently out of favor. if used, liver function should be closely monitored, and it is recommended that this regimen not be given to patients with preexisting liver disease (e.g., because of alcohol or viral hepatitis). because rifampin should not be used by subjects taking pis, this may also limit widespread application of the two-drug regimen. the same applies to combinations of isoniazid and rifampin taken for at least months, which are also effective in hivnegative individuals. alternate protocols also exist for subjects thought to be resistant to first-line prophylactic agents. these have not been widely clinically evaluated. it is recommended that hiv-infected subjects who have had close contact with an active case of tuberculosis should also receive prophylaxis. there is little evidence to suggest that anergy confers an increased risk for development of clinical disease. however, patients who have not had bcg, have a negative skin test, and have started haart may benefit from regular skin tests, because some studies suggest that cutaneous responses may return with increasing cd counts, and that this may help in identifying newly infected individuals requiring prophylaxis. in populations where the prevalence of tuberculosis is low and bcg may be given during childhood or adolescence (e.g., the united kingdom), the value of ppd testing is more limited. here, an arbitrary cutoff of mm for tuberculin reactions is used to define who should receive preventive therapy. the introduction of immune-based blood tests that can accurately distinguish between bcg vaccination and tuberculosis infection may be helpful when screening for evidence of latent tuberculosis. the two currently available commercial tests use fairly specific cd -directed mycobacterial antigen responses with consequent production of detectable interferon-g. the need for reasonably intact cd function means that they may, in fact, be less useful in hiv infection, especially in those subjects with very low blood cd counts, who are possibly at greatest risk of developing active tuberculosis. secondary tuberculosis prophylaxis may be important, because studies indicate a high rate of relapse in endemic areas. here, no specific guidelines exist, although months of isoniazid and rifampin after a full treatment course shows a greatly reduced risk of relapse within the subsequent years. whether this is enough to prevent clinical disease (which may also arise from reinfection in areas of high tuberculosis prevalence) without concomitant antiretroviral therapy is unclear. in the developed world, secondary prophylaxis is usually not recommended. the use of haart also can reduce the risk of tuberculosis in endemic areas. work in south africa indicates that this is most beneficial in patients with advanced disease and leads to a reduction in rr of at least %. data from north america indicate that the prevention of disseminated mac infection has an effect on survival ( % reduction in mortality rate in subjects taking clarithromycin). the us guidelines advise prophylaxis with a macrolide (either clarithromycin, mg orally twice per day, or azithromycin, mg orally, once a week) in all hiv-infected individuals with blood cd counts > cells/ml. in europe, where the prevalence of disseminated mac infection is probably lower (perhaps because of previous bcg vaccination), this may be less relevant. here, surveillance cultures of blood may be more cost-effective in the at-risk hiv population with low cd counts. routine stool and sputum cultures probably do not add much to this strategy, because disseminated mac is much more common than isolated organ disease. single-agent prophylaxis may lead to antibiotic resistance. this does not seem to be reduced by the addition of a second drug (rifabutin) to the prophylactic regimen. the latter is now a second-line prophylactic agent, largely as a result of its rather worse protective effect and its adverse interaction profile with pis. as mentioned earlier, if an individual sustains a rise in cd count > cells/ml for > months, it is safe to discontinue prophylaxis. several clinical and laboratory features have prognostic significance in hiv-infected individuals with pcp (box - ). a severity score on the basis of the serum ldh levels, the a-ao , and the percentage of neutrophils in the bal fluid can predict survival reasonably accurately, with the highest scores indicating the worst outcome. other workers have shown that increased age (< years) leads to an increased mortality rate-in part as a result of late, "unsuspected" diagnosis. the overall mortality rate from an episode of pcp is approximately % and has not changed since the advent of haart. among individuals with access to haart, post-pcp survival has improved. in , the median survival after pcp was months. by , this had risen to months. the introduction of haart has led to a further improvement, with survival in the period up to year of months. in those without access to haart and/or prophylaxis, survival post-pcp, unfortunately, remains poor. in general, mortality from bacterial respiratory infection in hiv-infected individuals is similar to that seen in the general population. clinical and laboratory markers of disease severity that have been defined in the adult general population (e.g., those described in the ats or the bts guidelines for the management of community-acquired pneumonia in adults) apply to hiv-infected patients. these are confusion, raised respiratory rate, abnormal renal function, and low blood pressure. recurrent pneumonia is common (reported in up to % of cases) and may lead to chronic pulmonary disease (see earlier). although tuberculosis normally responds to standard multipledrug therapy, work from africa has highlighted the increased mortality rate in hiv-infected compared with non-hivinfected individuals. a relationship has also been described between mortality and declining blood cd count: hivinfected patients with cd counts < cells/ml have a mortality rate of % compared with % in those with cd counts from - cells/ml. compared with hiv-infected individuals without tuberculosis, the main effect on mortality is seen in patients with higher cd counts (> cells/ml), where the relative risk of death is three times that of the nontuberculous population. several case-controlled studies have indicated that in the absence of effective treatments, mac-infected patients have a reduced survival compared with blood cd level-matched control subjects (approximately months vs months, respectively). currently available treatment regimens may reduce this difference, although severe anemia seems to be an independent predictor of mortality. the presence of cmv in bal fluid also containing p. jirovecii has been related to outcome (see earlier). the mortality rate at and months after bronchoscopy is greater in those with cmv detected at bronchoscopy. however, cmv recovered as a sole pathogen does not impact on survival. the effect of antiretroviral therapy on opportunistic infections the introduction of haart, together with the wide availability of accurate methods of determining plasma rna viral load, has led to profound changes in both clinical practice and hiv outcome. although it is still the case that respiratory disease remains above non-hiv infected background levels, in particular, bacterial pneumonia, tb, and lung cancer are more common, despite apparently effective haart, in hiv-infected subjects. overall data indicate that clinical progression is rare in subjects who are able to adhere rigorously to at least % of their antiretroviral drug regimen. mortality rates have fallen by % for almost all conditions, and it seems that a damaged immune system can, to a clinically significant extent, be reconstituted for a period of at least several years. hence, clinicians need to consider not only opportunistic infection or malignancy within their diagnostic workup but also the effects of drug therapy itself. the side effect profile of haart (e.g., metabolic and mitochondrial toxicities, liver damage, and neuropsychiatric disorders), as well as the large number of drug-drug interactions, makes this a very complex area of management. the best example of this is hiv-related tuberculosis. here, not only is there overlapping toxicity and pharmacologic interaction, but iris is common. research is needed to address this area. studies should inform the decision on when to start haart in patients already on antituberculosis medication. other work needs to focus on understanding box - prognostic factors associated with poor outcome in pneumocystis jirovecii pneumonia on admission patient's age no previous knowledge of hiv status tachypnea (respiratory rate > /min) second or subsequent episode of pneumocystis jirovecii pneumonia poor oxygenation À pao < . kpa (< mmhg) or a-ao > . kpa (> mmhg) low serum albumin (< g/dl) low hemoglobin (< . g/dl) peripheral blood leukocytosis (> . Â /l) elevated serum lactate dehydrogenase levels (> iu/l) cd count < cells/ml marked chest radiographic abnormalitiesdiffuse bilateral interstitial infiltrates with or without alveolar consolidation medical comorbidity (e.g., pregnancy) at bronchoscopy . in bronchoalveolar lavage fluid detection of a. copathology cmv bacteria b. neutrophilia (> %) . detection of pulmonary kaposi sarcoma serum lactate dehydrogenase levels that remain elevated development of pneumothorax high apache ii score on admission to the icu need for mechanical ventilation why full pulmonary immunity is not restored. this may reflect abnormalities in the innate immune response, which is currently poorly described in hiv infection. despite the benefits of haart, it is likely that in the long term many patients will progress to severe disease. there is currently little research in this area. research should focus on correlating clinical and laboratory findings. an example of this would be assessing the risk of an individual for development of active tuberculosis. it is clear that much of the excess mortality in hiv-tuberculosis coinfection occurs early in hiv infection. thus, if tests can be devised that indicate who has latent tuberculosis infection (and who is, therefore, most likely to have clinical disease develop), steps can be taken to prevent illness. as discussed previously, immune-based tests have shown promise in immunocompetent individuals with tuberculosis infection. if these can be refined to work consistently in patients with hiv infection at a reasonable cost, there is the possibility of targeting those at risk of future tuberculosis, or of tuberculosis "unmasking" after starting haart. the other role for a test such as this would be in rapid diagnosis of active tuberculosis. it is common to be faced with a patient who has nonspecific symptoms and a wide differential diagnosis. often treatment is multiple and empirical. a quantitative test would help resolve some of these dilemmas by indicating the chance of the condition being caused by a particular disease. an example would be the patient from an endemic tuberculosis area, with low cd counts, who has both pulmonary and central nervous system disease. is this tuberculosis, toxoplasmosis, cryptococcosis, or viral or bacterial infection? any such test for tuberculosis would also have to distinguish between the different states of old (treated), old (inactive), old (latent), and active. although not insurmountable, at present, this is not possible. rapid diagnostic assays that assess organism viability are also important. if a clinician can receive early feedback on whether treatment is producing a suitable killing effect, therapy can be tailored to the individual. this enables regimens to be "dose adjusted" as needed and removes the element of concern that is often present when patients are slow to respond. examples of this would be in the treatment of pcp or mycobacterial disease. the frequency of bacterial infection (often recurrent) with its attendant sequelae makes effective strategies for vaccination an important priority. it is uncertain why there is a differential response to vaccination; even in the united states, african americans do not seem to derive the same benefit as whites. this needs further research, together with more emphasis on identifying the local immune response present in the lung in such individuals. bacterial infections may be clinically indistinguishable from other pathogens, and only two thirds of all respiratory infections are formally diagnosed. there is a need for improved methods to assist with this. the use of rapid antigen tests may be one way forward. this is especially so given the high incidence of (potentially fatal) bacteremia present in such populations. for maximum benefit, this needs to use a system that is simple and cheap, and hence suitable to both resourcerich and resource-poor countries. m. tuberculosis is globally the most important hiv-related pathogen. strategies of control and prevention are vital to ensure that millions of people do not become coinfected and that those who are do not go on to have clinical disease develop. rapid diagnostics are critical. the encouraging reports of the simple and cheap method of mods to both diagnose tuberculosis and then provide resistance data in field settings (see earlier) argues for large-scale roll out and evaluation. beyond public health measures, such as dot, fixed-dose combination drugs, case management, and education, research needs to improve on current drug therapy. long-acting preparations such as rifapentine show promise but, as the problem with rifampicin monoresistance demonstrates, there is still much work to be done. for the first time in many years, there are several antimycobacterial drugs that are in various stages of clinical trials. all are promising, and several have novel mechanisms of action. the global alliance and the who "stop tb" campaigns have been crucial in this regard. the fluoroquinolones, moxifloxacin and gatifloxacin, are closest to the market. they are potent drugs with considerable ability both to kill and also sterilize mycobacteria-infected sites. trials of treatment-shortening regimens are ongoing worldwide. vaccination against m. tuberculosis with bcg has understandably not been widely used in an immunosuppressed hiv-infected population. however, a safe vaccine may be the only affordable way of protecting large parts of the world from tuberculosis. so far there seems to be more success in vaccines to either enhance or replace the primary protective effects of bcg. the use of immunotherapy (e.g., with heat-killed mycobacterium vaccae) in combination with chemotherapy has been disappointing in clinical trials. newer methods of diagnosis (e.g., pcr tests on saliva) may prove invaluable for quick and easy disease confirmation, although their applicability to routine samples needs further evaluation. p. jirovecii prophylaxis was the first important hiv treatment widely available. however, despite the efficacy of tmp/smx, compliance remains a problem. regimens that use a gradual increase in dosage when starting prophylaxis may help. one concern with widespread use of prophylaxis is that resistance will start to occur to tmp/smx. reports have indicated that there are mutations in the p. jirovecii dihydropteroate synthase gene that confer resistance. these seem to be increasing over time, although they do not seem to be present in many patients who fail treatment for pcp with tmp/smx. the implications of this are uncertain but could include a greater likelihood of treatment failure and the possibility of worsening patterns of global bacterial drug resistance. hiv-infected populations in the developed world have high rates of smoking. the evidence that this is harmful above those effects seen in the general population continues to accrue. the accelerated course of both obstructive lung disease and cancer, together with the increased risk of respiratory infection in smokers, persuasively argues the case for targeted smoking cessation. that hiv infection and haart have profound (and probably negative) effects on blood lipids and insulin resistance further support the need to reduce smoking rates in this population. it seems that we are starting to see increased rates of cardiovascular disease in this now aging population. the natural history of hiv-related respiratory disease continues to evolve. haart and newer therapeutic strategies have made a significant impact on morbidity and mortality. yet individuals continue to become hiv infected, progress, and die from an ever-expanding range of conditions. p. jirovecii remains the most common aids-defining event in the developed world, whereas m. tuberculosis is globally the most common cause of death. bacterial respiratory infection is not far behind. given the huge number of individuals with hiv infection, the only effective way to manage this disease is to find simple ways of treating hiv itself, and thus contain the worst ravages of this illness. treating opportunistic infections among hiv-infected adults and adolescents revised recommendations for hiv testing of adults, adolescents, and pregnant women in healthcare settings treatment of hiv-related tuberculosis in the era of effective antiretroviral therapy treatment and prophylaxis of pneumocystis carinii pneumonia immune reconstitution disease associated with mycobacterial infections in hiv-infected individuals starting antiretroviral therapy immune reconstitution inflammatory syndrome in hiv guidelines for preventing opportunistic infections among hiv-infected persons- on behalf of the bhiva guidelines writing committee pneumocystis and trypanosoma cruzi: nomenclature and typifications oxford: blackwell scientific aids and respiratory medicine key: cord- -pta nzz authors: murphy, caitlin n.; fowler, randal; balada-llasat, joan miquel; carroll, amanda; stone, hanna; akerele, oluseun; buchan, blake; windham, sam; hopp, amanda; ronen, shira; relich, ryan f.; buckner, rebecca; warren, del a.; humphries, romney; campeau, shelly; huse, holly; chandrasekaran, suki; leber, amy; everhart, kathy; harrington, amanda; kwong, christina; bonwit, andrew; dien bard, jennifer; naccache, samia; zimmerman, cynthia; jones, barbara; rindlisbacher, cory; buccambuso, maggie; clark, angela; rogatcheva, margarita; graue, corrin; bourzac, kevin m. title: multicenter evaluation of the biofire filmarray pneumonia/pneumonia plus panel for detection and quantification of agents of lower respiratory tract infection date: - - journal: j clin microbiol doi: . /jcm. - sha: doc_id: cord_uid: pta nzz the ability to provide timely identification of the causative agents of lower respiratory tract infections can promote better patient outcomes and support antimicrobial stewardship efforts. current diagnostic testing options include culture, molecular testing, and antigen detection. these methods may require collection of various specimens, involve extensive sample treatment, and can suffer from low sensitivity and long turnaround times. this study assessed the performance of the biofire filmarray pneumonia panel (pn panel) and pneumonia plus panel (pnplus panel), an fda-cleared sample-to-answer assay that enables the detection of viruses, atypical bacteria, bacteria, and antimicrobial resistance marker genes from lower respiratory tract specimens (sputum and bronchoalveolar lavage [bal] fluid). semiquantitative results are also provided for the bacterial targets. this paper describes selected analytical and clinical studies that were conducted to evaluate performance of the panel for regulatory clearance. prospectively collected respiratory specimens ( bal and sputum specimens) evaluated with the pn panel were also tested by quantitative reference culture and molecular methods for comparison. the pn panel showed a sensitivity of % for / etiologic targets using bal specimens and for / using sputum specimens. all other targets had sensitivities of ≥ % or were unable to be calculated due to low prevalence in the study population. specificity for all targets was ≥ . %, with many false-positive results compared to culture that were confirmed by alternative molecular methods. appropriate adoption of this test could provide actionable diagnostic information that is anticipated to impact patient care and antimicrobial stewardship decisions. ) and is a leading cause of hospital and emergency room visits. the highest morbidity and mortality of these illnesses are frequently seen in the elderly, children Ͻ years of age, and the immunocompromised ( ) . pneumonia-like illness is also a frequent hospital-acquired infection that can result in increased mortality and unnecessary economic burden ( ) . bacteria and viruses are the most common etiologies of lower respiratory tract infections. patients with viral pneumonia may be managed differently than those with bacterial infections, but due to similarities in clinical presentation and symptomatology, it is not possible to distinguish viral from bacterial infections without the aid of laboratory diagnostic testing. rapid diagnostics for specific entities (streptococcus pneumoniae and respiratory syncytial virus [rsv] ) and host markers (procalcitonin) exist for the detection of common viral and bacterial illness and/or to aid in distinguishing bacterial from viral infections ( ) ( ) ( ) . these methods alone are frequently inadequate as a means to diagnose and treat pneumonia ( , ) . rapid resolution of the etiology of lower respiratory tract infections can aid in the ability to ensure that appropriate antimicrobial therapy is initiated and that patients are put on the appropriate infection control precautions and to prevent unnecessary downstream testing. in adult populations, broad-spectrum antibiotics are often initiated before bacterial culture results are available, if there is suspicion of bacterial pneumonia or if the patient requires icu admission ( ) . in children, viral entities are the most frequent cause of pneumonia, and directed antiviral therapy is recommended for severely ill patients ( ) . rapid detection of the causative agent of respiratory infection, coupled with detection of prominent markers of antibiotic resistance, can aid in limiting unnecessary broad-spectrum antimicrobial treatment. the biofire filmarray pneumonia panel (pn panel) and pneumonia plus panel (pnplus panel) (biofire diagnostics, llc, salt lake city, ut) were designed to provide a means of rapidly detecting nucleic acids from common agents of community-and hospital-acquired lower respiratory tract infections ( table ). the panel integrates nucleic acid extraction, reverse transcription, and nested multiplex pcr amplification for (pn panel) or (pnplus panel) viruses, bacteria (including atypical bacteria associated with community-acquired pneumonia), and antimicrobial resistance (amr) genes. the pn panel and pnplus panel test reagents are identical, with results for middle east respiratory syndrome coronavirus (mers-cov) masked by the software for the pn panel version; for simplicity, the tests are referred to collectively as the pn panel throughout this paper except where a distinction is required. the device is intended for use with sputum-like specimens (expectorated or induced sputum and endotracheal aspirates [eta] ) and bronchoalveolar lavage (bal) specimens tested directly, without pretreatment. in addition to nucleic acid detection, the panel is able to provide a semiquantitative estimate of abundance for of the bacterial targets (reported in log increments from to genomic copies/ml). all testing is done in the closed sample-to-answer filmarray system, which provides automated analysis and results in about min. here, we report on studies performed to characterize the linearity and accuracy of the semiquantitative results provided for the bacteria detected by the pn panel as well as a multicenter prospective study, where the performance of the panel was evaluated in comparison to several reference methods that included conventional and quantitative culture and molecular detection. contrived samples for linearity and accuracy validation. dilutions of contrived bal samples containing cultured bacterial isolates in a matrix of sterile physiological saline and ng/l human genomic dna were tested repeatedly with the pn panel ( replicates) to assess both the linearity and accuracy of the test's semiquantitative bin results. each bacterium was tested at six concentrations in -log intervals extending above and below the reportable range of the panel (Ͻ . through Ն copies/ml). the reference or input concentration of bacterial genomic dna (copies per milliliter) in each contrived sample was determined by digital pcr (dpcr). the nucleic acid quantification method implemented mechanical and chemical lysis of each cultured bacterium (bead-beating on the disruptor genie [scientific industries] at approximately , rpm for min and magna pure bacterial lysis/binding buffer) followed by total nucleic acid extraction and purification using the roche magna pure lc . platform (roche diagnostics, indianapolis, in). extracts were quantified by dpcr (quantstudio d digital in the study. a waiver of the requirement for informed consent was obtained from the institutional review board (irb) at each study site for the use of residual specimens and in order to collect subject information from the medical records. clinical and demographic data were collected, including hospitalization status at the time of specimen collection, the results of the clinician-ordered soc culture, subject sex, and subject age category. sites were instructed to enroll specimens in the morning as the first study activity of the day so that all specimen aliquoting, shipping, freezing, and pn panel testing were performed in temporal proximity. specimens were coded or pseudonymized by the study enroller, thoroughly mixed by vortexing, and then pipetted into various aliquots for testing. one aliquot was used for testing on-site with the pn panel. an additional aliquot was shipped overnight at refrigeration temperature to a central reference laboratory (mriglobal, palm bay, fl) for reference culture, and finally, several aliquots were immediately frozen for molecular comparator testing. pn panel testing. this study was conducted with an investigational-use-only (iuo) version of the pn panel that is identical to the commercial (i.e., fda-cleared, ce-marked) in vitro diagnostic (ivd) version. all specimen handling occurred in a biosafety cabinet with operators wearing appropriate personal protective equipment, preparing one specimen at a time, and cleaning between specimens, all according to the manufacturer's instructions ( ) . in contrast to other biofire filmarray test panels, which use a transfer pipette for specimen loading, specimens are introduced into the pn panel test with a provided flocked swab. this facilitates recovery of organisms from viscous lower respiratory tract specimens. briefly, the native specimen was collected on the provided sample transfer swab (approximately l) and placed in sample buffer within the filmarray injection vial (faiv). the sample swab was broken off inside the faiv at a prescored breakpoint. after the lid was closed, the faiv was gently inverted three times to facilitate organism release, and then the contents were injected into the pn panel pouch before testing with the filmarray instrument. the pn panel test consists of automated nucleic acid extraction, reverse transcription, nucleic acid amplification, and automated results analysis in approximately min per run (i.e., per specimen). if either of two internal controls fails, the software automatically provides a a levels of , . , and are considered low, medium, and high, respectively. b p. mirabilis in one bal specimen was reported as "not detected" by the biofire pn panel. result of "invalid" for all panel analytes. viruses and atypical bacteria are reported qualitatively as "detected" or "not detected" (an "equivocal" result is also possible for mers-cov on the pnplus panel). the amr genes are also reported qualitatively ("detected" or "not detected"), but only if one or more applicable bacteria (i.e., potential carriers of the amr gene) are also detected in the sample (table , footnotes c and d); if no applicable bacteria are detected, the amr gene results are reported as "n/a" (not applicable) . for bacterial targets, the biofire pn panel calculates an approximate quantity of the gene target (i.e., bacterial dna, in copies per milliliter) based on real-time amplification curves for the bacterial assays relative to a quantified internal reference standard manufactured into each pn panel test cartridge. the assays are designed to amplify genes that are present in single copies within the chromosome of the target bacterium and thus to estimate a concentration of targeted bacterial genome equivalents in the specimen. the calculated value is rounded to the nearest n value and reported as a bin result ( , , , or Ն genomic copies/ml). assays with no measurable amplification or a calculated value below . ( , ) copies/ml are considered negative and reported as "not detected." comparator testing. (i) standard-of-care culture. all eight study sites followed their own standard procedures to determine soc culture results, independent of the study. while the methods for culture of lower respiratory tract specimens are relatively standardized, each site (and sometimes technicians within a site) had variation with respect to whether and how organisms were reported ( ) . results were obtained from chart review of subject medical information. (ii) quantitative reference culture. a central reference laboratory (mriglobal, palm bay, fl) was used to perform quantitative reference culture (qrefcx). this approach was similar to the method that the study sites use for routine standard-of-care (soc) culture; however, different sites' soc protocols varied. the reference lab was used in order to standardize the plating protocol and results and, in particular, to ensure that quantitative results were obtained over the reportable range of the pn panel for both specimen types. aliquots of enrolled specimens were shipped overnight at refrigeration temperature (on ice) to the central reference laboratory. specimens were excluded if they did not arrive at the reference lab with sufficient time to be processed for culture within one calendar day of enrollment or if they were no longer at refrigeration temperature upon arrival. bal and sputum specimens were treated the same except that sputum specimens were pretreated with an equal volume of snotbuster (copan, murrieta, ca) mucolytic reagent to reduce viscosity before plating. specimens were streaked onto four different media (blood agar, chocolate agar, macconkey agar, and columbia colistin-nalidixic acid [cna] agar) at four different concentrations: l and l of both undiluted and : -diluted specimen. plates were incubated at °c and inspected for growth at and h. quantity was determined by counting colonies of each unique morphology on the plate type with the most robust growth of that morphology and at the dilution with to colonies of that morphology. if an organism was observed on multiple plates, the highest quantification value was used. identification was described first by colony morphology and then confirmed by vitek id (biomérieux, durham, nc) following isolation and subculturing. vitek was also used for phenotypic antimicrobial susceptibility testing (ast). glycerol stocks of relevant bacterial isolates were prepared for molecular amr gene testing and discrepancy investigation. (iii) real-time pcr and sequencing. total nucleic acids were extracted from clinical specimens using a magna pure lc . instrument. atypical bacteria and viruses were tested with two well-validated nested pcr assays; amr genes were tested with a single assay. whenever possible, the comparator pcr assays targeted different genes (or different regions of the same gene) than are targeted by the pn panel assays. assays were designed to generate amplicons that would provide sufficient sequence information for conclusive analyte identification (between and bp). a sequence-confirmed positive result from either assay was considered positive for a given analyte. validation testing demonstrated that most assays (at least one or both per analyte in both bal and sputum sample types) had a limit of detection (lod) that was within at least -fold that of the pn panel, which was considered "equivalent" sensitivity. all specimens were assumed to be negative for mers-cov, as it was not circulating in the united states during the time of enrollment for the study; no comparator testing was performed for this analyte. results and discrepant analysis. a pn panel result was considered a true positive (tp) or true negative (tn) when it agreed with the result from the comparator method. discrepant analysis was performed when results were discordant, i.e., false-positive (fp) or false-negative (fn) results. when sufficient specimen volume was available, discordant specimens were investigated using a combination of retesting with the pn panel or comparator methods as well as testing with additional, independent molecular assays. note that the performance data for positive percent agreement (ppa) and negative percent agreement (npa) presented in this paper consist of unresolved data as presented in the package insert for the commercial test; discrepancy investigation is provided but was not used to recalculate performance data. statistical analysis. the exact binomial two-sided % confidence intervals ( % ci) were calculated for performance measures according to the wilson score method ( ) . linearity and accuracy of pn panel semiquantitative bin results for bacteria. each pn panel bacterial assay was designed to be efficient and linear and to provide accurate bin results within Ϯ . log copies/ml of the input concentration over a reportable range of to Ͼ copies/ml. the linearity and accuracy of the assays were validated by testing a -log dilution series of contrived samples containing each bacterium detected by the panel. results for staphylococcus aureus and klebsiella aerogenes are shown in fig. as representative of gram-positive and gram-negative organisms, respectively; additional data for all other bacteria can be found in the product instructions for use ( ) . each of the samples at six concentrations was tested repeatedly ( pouches) and the bin results of the test (in copies per milliliter) were compared to the input concentration (also in copies per milliliter) of the sample. contrived samples containing staphylococcus aureus at input concentrations of , , , , , and copies/ml (representing the "middle" of the bin) were tested with the pn panel, and s. aureus was detected in % ( concentrations within the reportable range of copies/ml and higher (fig. a) . over the dilution series, the semiquantitative bin result changed linearly, in direct proportion to the change in sample input concentration (e.g., an increase in concentration of log copies/ml generated a change in bin result equivalent to log copies/ml). in addition, the semiquantitative bin result reflected the sample input concentration within the stated Ϯ . -log -copies/ml accuracy of the panel. for example, the sample input concentration of copies/ml has an expected accuracy range of . to . , and an accurate bin result of copies/ml was reported by the pn panel for % of the sample replicates tested. contrived samples containing k. aerogenes were tested at input concentrations of . , . , . , . , . , and . copies/ml (representing the "edge" of the bin), and k. aerogenes was detected in % ( / ) of all replicates at a concentration of . copies/ml and higher (fig. b) . the semiquantitative bin result changed linearly, in direct proportion to the change in sample concentration, though at each concentration except the highest two bins, results were reported in variable proportions over the replicates. although more than one bin result was reported in different replicates of the same input concentration, each bin result was accurate relative to the input concentration within Ϯ . log copies/ml. for example, the sample input concentration of . copies/ml has an accuracy range of . to . copies/ml (spanning two bins). the pn panel provided an accurate bin result of copies/ml in . % of the replicates tested at this concentration with an equally accurate bin result of copies/ml for the remaining . % of the sample replicates tested. semiquantification in contrived polymicrobial specimens. the organisms are reported at a semiquantitative level, and thus, the accuracy of the expected relative rank order among contrived polymicrobial specimens (low, medium, and high) was tested. in ( %) contrived sputum specimens and in of ( . %) bal specimens (table ) , the correct relative rank was observed. four specimens in bal sample set that were spiked with e. cloacae at a medium level of . genomic copies/ml were reported by the pn panel as "detected" at Ն (high) instead of the expected level of . all other organisms in these four samples were reported at the correct level, and this organism was reported at the correct level in all specimens of the corresponding sputum sample set, set . one additional specimen in bal sample set that was spiked with p. mirabilis at genomic copies/ml was unexpectedly negative, but the other two organisms in the specimen were reported correctly. all other results for p. mirabilis in all other samples were reported correctly. clinical demographics. a total of bal specimens ( bal and mini-bal specimens) and sputum specimens ( sputum specimens and eta) were collected for the prospective clinical study from eight u.s. clinical sites. fifty-eight bal and sputum specimens were excluded after enrollment. the most common reasons for specimen exclusion was that reference culture could not be performed within the required time frame (as described in materials and methods). sex, age, and patient care setting (hospitalized, outpatient, or emergency department [ed]) were recorded for all subjects from whom specimens were enrolled. the clinical demographics associated with the , valid enrolled specimens are presented in table test performance and summary of the pn panel. in the prospective clinical evaluation, a total of , of , pn panel test runs ( bal and sputum specimens) were completed on the first attempt, for an overall instrument success rate of . %. of the , completed runs, , ( . %) produced a valid result (i.e., successful pouch controls). twenty-eight of the specimens with control failures had sufficient volume for retesting and were able to be retested within study-defined time interval (without specimen dilution or manipulation); produced a valid result on the single retest. the pouch controls failed a second time for the remaining three specimens, and there was no further specimen volume for testing. of the valid runs, the pn panel detected at least one analyte in of bal specimens and in of sputum specimens for an overall positivity rate of . and . %, respectively (table ). codetections were observed in . % ( / ) of bal specimens and . % ( / ) of sputum specimens. the most commonly detected analytes were staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae, and human rhinovirus/enterovirus (hrv/ev), which were found in ( %), ( . %), ( . %), and ( . %) specimens, respectively. all other analytes were detected in fewer than ( . %) specimens. the overall prevalence of each analyte stratified by collection location is shown in table . qualitative analysis of typical bacteria. the performance characteristics of the pn panel for semiquantifiable bacterial targets compared to the reference method of qrefcx performed at the central lab are presented in table . a specimen was considered positive for a particular organism by qrefcx when it was recovered and enumerated at a level greater than , ( . ) cfu/ml approximated using dilution plating, which is equal to or greater than the pn panel reporting threshold of . genomic copies/ml. the overall sensitivity for sputum samples ranged from % to %, and that for bal specimens ranged from . % to %. sensitivity for a. calcoaceticus-a. baumannii, moraxella catarrhalis, and streptococcus agalactiae could not be calculated for bal specimens due to limited detections by the qrefcx comparator method. specificity for all analytes in both specimen types ranged from . % to . %. compared to a quantitative reference culture, false-negative results were uncommon, with no more than observed for any organism and total among the , specimens tested. comparatively, false-positive results were relatively common in both specimen types. the highest rates of false-positive detections were seen for the organisms most frequently detected: total for both s. aureus and h. influenzae, for m. catarrhalis, and for p. aeruginosa. discrepancies between positive detection by pn panel and negative qrefcx culture report were evaluated by first determining if the organism was reported as negative because it was enumerated below the threshold of Ͻ . ( , ) cfu/ml set for culture. if discrepancies remained unresolved, the results of an independent molecular assay were considered. finally, if discrepancies remained, the results from soc testing at the individual sites were considered. results of discrepancy analysis are shown in table . a total of discrepant false-positive results were observed between the pn panel and comparator qrefcx. a quarter ( . %; / ) of the discrepancies between the pn panel and qrefcx were resolved as the organism being present but enumerated below the reference culture cutoff of . cfu/ml. an additional . % ( / ) were resolved using the results of an alternative molecular method or by evaluating the results of soc culture. among specimens with false-negative results, evidence of the target organism was found in specimens by molecular testing ( specimens) or soc culture ( specimen); the false-negative results were attributed to low levels of organism in the specimen, i.e., at or below the pn panel reporting cutoff. sequencing of bacterial isolates recovered from five remaining false-negative specimens indicated misidentification by the reference lab performing qrefcx (one a. baumannii isolate sequenced as pseudomonas fluorescens, one h. influenzae isolate sequenced as haemophilus haemolyticus, one k. aerogenes isolate sequenced as hafnia paralvei, and two p. aeruginosa isolates sequenced as pseudomonas denitrificans and pseudomonas fluorescens). investigation of the final false-negative k. pneumoniae result uncovered evidence of a specimen swap or paperwork error. following discrepancy testing and analysis, only three false positives remained unresolved. no evidence of nonspecific amplification was observed for the pn panel. an additional qualitative analysis of the pn panel semiquantitative results for bacteria was performed by comparing them to soc culture results (table ). in this analysis, an organism was considered positive by soc if a result for the particular bacterial analyte was entered in the subject's medical record, regardless of any quantity information that may have been indicated. while specificity by this method is similar to that of the qrefcx culture method, sensitivity is lower for some analytes. this was attributed to the fact that the analysis considered an analyte positive by soc if it was reported at any level. while some organisms were reported in subject medical records with a numerical quantity, most were reported with qualitative descriptions such as "few," "most abundant," " ϩ," etc., and therefore, this information could not uniformly be converted to a numeric value equivalent to the pn panel reporting threshold. an investigation of pn panel false-negative results relative to soc revealed that the majority ( ; . %) had been reported as being present at a low level (i.e., "few") and may have been present below the pn panel cutoff. thirteen ( %) were reported at higher levels, and ( . %) could not be categorized because the reported quantities were described in relative terms (e.g., "listed first" or "least of three"). quantitative analysis of typical bacteria. pn panel semiquantitative results were compared to qrefcx results (table ) using the following analysis. qrefcx results for each organism were stratified into -log ranges (e.g., to Ͻ , to Ͻ , etc.) ( table ). the pn panel bin result for a particular analyte was considered concordant if the reported bin value was at either end of that range (e.g., a qrefcx value of , cfu/ml, or . ϫ , which falls between and , was concordant with a pn panel bin result of either or ). concordance was low for qrefcx values below cfu/ml, with overall values ranging from . % to . % for both specimen types ( table , "ϭ" columns). however, when qrefcx values were above , pn panel concordance was . % to % for both specimen types. when discrepant results were examined for a particular concentration range, there were very few instances where the pn panel result was "not detected" or was a value lower than that from a spu, sputum. b reported organism quantity in subject medical record. "few" corresponds to values of Ͻ , cfu/ml and the descriptions "few," " ϩ," "light growth," "rare," and " colony"; "mod" corresponds to values of , to Ͻ , cfu/ml and the descriptions "moderate," " ϩ," and " ϩ"; "many" corresponds to values of Ն , and the descriptions "many," "heavy growth," and " ϩ." uq, unable to quantify (quantity was given in relative terms, e.g., "listed first" or "least of three"). c bq, organism present in qrefcx but enumerated below the quantification threshold of qrefcx ( table , "nd" and "Ͻ" columns). however, the pn panel reported organism levels higher than the qrefcx range for . to . % of specimens with qrefcx values below ( table , "Ͼ" columns). performance was similar for all organisms. s. aureus meca/mecc and mrej determinants. when s. aureus is detected on the pn panel, it is accompanied by a result for the detection of meca and mecc. these genes encode a penicillin-binding protein (pbp a) that has low affinity for beta-lactams and are carried on a chromosomally integrated mobile genetic element called the staphylococcal cassette chromosome mec (sccmec), which may be found in many staphylococcus spp. to distinguish between methicillin-resistant s. aureus (mrsa) or codetection of methicillin-sensitive s. aureus (mssa) and another staphylococcus sp. carrying the sccmec cassette and meca/mecc, the pn panel contains an additional assay that amplifies the sccmec right-extremity junction (mrej), which links the sccmec cassette to the s. aureus genome and indicates mrsa. s. aureus was detected in bal and sputum samples. the pn panel meca/mecc and mrej "detected" results for these specimens were compared to results of molecular testing performed directly from the specimen, with ppa and npa of . % and . % for bal and . % and . % for sputum, respectively ( table ). investigation of the false-positive and false-negative specimens using independent molecular methods found evidence of meca/mecc and mrej in of them (table ; one specimen could not be investigated due to lack of remaining volume). a review of ast testing performed on s. aureus isolates recovered by soc and qrefcx methods (data not shown) revealed that many of the specimens with discrepant results were polymicrobial with both mrsa and mssa. some specimens were polymicrobial with other methicillin-resistant staphylococcus spp. (i.e., organisms carrying meca/mecc) and an mssa isolate which may have carried an empty sccmec cassette (and thus was positive for mrej and meca/mecc but was not mrsa). when these different organisms are present together at near-lod levels in polymicrobial specimens, differential detection by the pn panel and reference methods (including phenotypic ast) leads to discordant results ( ) . the mrej sequence from one falsenegative specimen was found to contain a sequence that is nonreactive to the pn panel mrej primers; this limitation is noted in the product instructions for use ( ) . carbapenemase and extended-spectrum beta-lactamase amr performance. the pn panel includes assays for six amr genes associated with carbapenem and extended-spectrum beta-lactam resistance that are reported for select gram-negative bacteria. these genes are reported as "n/a" if no applicable host organism is detected in the specimen (table , footnotes b, c, and d) . ctx-m and kpc were the most commonly detected amr targets in both bal and sputum samples (table ). vim was detected in two sputum samples, ndm was detected in one bal specimen, and imp and oxa- -like genes were not detected. the comparator method for amr gene a reported only when an applicable host organism is also detected by the biofire pn panel (see table , footnotes b, c, and d). performance was an independent molecular method performed on the specimen (a comparison of pn panel amr gene detection to phenotypic ast of recovered isolates may be found in the pn panel instructions for use [ ] ; however, this method was not used as a primary comparator because the pn panel detected more organisms than were recovered by culture [ table ] and also because phenotypic antimicrobial susceptibility may be conferred by mechanisms other than the genes reported by the pn panel, thus confounding interpretation of such results). kpc detection had a performance of % ppa in both sample types, with % npa in sputum and . % npa in bal. ctx-m detection had a ppa of . % in bals and % in sputum samples and npa of % in bals and . % in sputum samples. of two vim detections in sputum specimens, one was true positive ( % ppa) and one false positive (resulting in . % npa). there was one ndm detection in bal, but it was a false positive ( . % npa); the comparator method also detected a single ndm, but this was not observed by the pn panel and was considered to be a false negative ( % ppa). discrepancy investigation with independent molecular methods found evidence of the amr gene in several of the discrepant positive and negative results (resolved true positives and confirmed false negatives) (table ) , suggesting analyte presence near the lod of both the pn panel and comparator assays. as overall prevalence of these resistance gene markers was low in the study population, contrived specimens were utilized to further demonstrate the positive and negative percent agreement of the resistance targets as described in the product instructions for use ( ) . analysis of viruses and atypical bacteria. the overall performance of atypical bacterial and viral targets on the pn panel is summarized in table . the ppa, npa, and % ci were calculated compared to comparator methods of pcr and sequencing. ppa for mers-cov could not be calculated, as no detections occurred during the course of this study; npa was %. the ppa for / targets was % for both bal and sputum. the lowest ppa for sputum was . % for adenovirus, with discordant results observed among all age groups; ppa for adenovirus was % in bal. for bal specimens, the lowest observed ppa was . % for coronavirus. atypical bacterial detections were rare overall, with the most frequent coming from mycoplasma pneumoniae, which demonstrated ppa and npa of . % to %. discordant results were attributed in most cases to low levels of analyte, i.e., at or near the lod; investigation with independent molecular assays found evidence of analyte presence in the majority of false-positive and nearly all false-negative specimens (table ) . lower respiratory tract infections can be caused by a wide range of pathogens. commonly, multiple diagnostic tests, including culture, molecular detection, and antigen detection, may be ordered to aid in the diagnosis of these infections. while awaiting the results of diagnostic testing, many patients are placed on broad-spectrum antibiotic therapy. in the absence of a clear diagnosis, antibiotic de-escalation may be delayed or rarely initiated. furthermore, it is estimated that % of cases of communityacquired pneumonia (cap) have no identified etiological cause ( ) . due to the insensitivity of culture, the infectious disease society of america (idsa) does not recommend culture of lower respiratory tract specimens for ambulatory patients with cap, owing to the low yield of culture and resulting minimal impact on patient care ( ) . culture remains the recommendation for patients with severe cap and for hospitalized patients with pneumonia. molecular methods for a variety of infectious processes have shown a clear increase in sensitivity and rapid turnaround times ( ) ( ) ( ) . this evaluation of the pn panel demonstrates the performance of this multiplex ivd test in selected analytical validation studies and a large prospective set of residual samples collected from a geographically and demographically diverse patient population. with the exception of a few targets that were not circulating in the population during the study period (e.g., mers-cov, chlamydia pneumoniae, and some amr genes), considerable numbers of most analytes were detected in both specimen types, allowing the determination of sensitivity/ppa and specificity/npa. the panel detects routinely encountered gram-positive and gram-negative pathogens. the sensitivity of this assay was Ͼ % for of these analytes in both bal and sputum specimens. sensitivities for the other five organisms ranged from % to . %. specificity for all targets in both specimen types was Ͼ %. the most challenging observation from these data is the discrepancy between the pn panel and culture for the detection and quantification of bacterial analytes. as shown in table , the pn panel demonstrated a lower specificity for bacterial analytes that were commonly detected (s. aureus and p. aeruginosa) than qrefcx. this finding correlates with those for other diagnostic assays that have been developed for the detection of lower respiratory tract pathogens ( ) , highlighting the increased sensitivity of molecular methods compared to culture for common pathogens. this is attributed to multiple factors. while culture remains the gold standard in the diagnosis of bacterial respiratory tract infections, it may be difficult to accurately recover all pathogens in clinical samples, as the organisms are in a complex matrix. in addition, culture results would be more affected by host immune response and prior antibiotic usage. culture is also subject to the criteria of each laboratory and to interpretation by the technologists examining those cultures. the panel is more robust against variability than could be attributed to the sample matrix, different techniques among laboratories, and recovery of more fastidious organisms. a potential drawback of molecular methods is the detection of nonviable organisms, but that may aid in the de-escalation of antibiotics in the absence of organism detection by culture in patients with prior antibiotic exposure. the pn panel was shown to reliably detect and quantify bacterial genomes ( table ) and was also shown to be able to detect the relative abundance of each target in contrived polymicrobial specimens (table ). further work is needed to determine if detection of organisms at low abundances in the pn panel that are not identified in culture is significant for patient outcomes. preliminary work done concurrently during this trial demonstrated the potential use of this panel as a diagnostic tool ( ) . a challenge of interpretation of respiratory cultures or results from molecular diagnostics like the pn panel is determining if the organisms detected are clinically significant. many clinically significant organisms may be normal flora of the oropharyngeal tract, particularly when they are present in a lower abundance. in the culture of lower respiratory tract specimens, it is important to report significant amounts of pathogens from sputum (often defined as presence of the organism in the second or third quadrant) or Ն cfu in bal specimens. previous studies have shown that quantitative pcr can be a means to differentiate commensalism from pathogenicity by looking at the nucleic acid burden ( ) . to promote adherence to current idsa recommendations, the pn panel reports only organisms that are detected at Ͼ . copies/ml. it then places the positive results into semiquantitative bins of , , , and Ն . in culture, it may be difficult to find significant organisms present in lower, but still clinically relevant, amounts in the presence of large numbers of other pathogenic or commensal organisms. the pn panel demonstrated that detection of organisms near the limit of detection was not influenced by the presence of a high burden of other organisms ( table ) . the data collected in this prospective study demonstrate that the pn panel is sensitive for the detection of bacterial analytes, as only a limited number of false negatives were observed when the pn panel was compared to qrefcx or soc (table ). this indicates that the panel cutoff of . genomes/ml is appropriate. the false negatives were attributed to organisms present in numbers below the lowest pn panel bin due to misidentifications at the central reference lab. the pn panel is additionally able to provide preliminary indication of potential antimicrobial susceptibility data for some commonly encountered pathogens via detection of selected amr genes. detection of meca/mecc in conjunction with mrej was shown to have high ppa and npa with an independent molecular method, ranging from . % to . %. the panel is also able to detect ctx-m-type extended-spectrum beta-lactamases (esbls). since the emergence of ctx-m-type esbls in the s, these enzymes have become the most prevalent type of esbl in a variety of settings throughout the world ( ) ( ) ( ) . ctx-m-type esbls are most prominent in e. coli and klebsiella spp.; e. coli strains carrying ctx-m are prominent causes of community-onset urinary tract and bloodstream infections. ctx-m results are reported when any member of the family enterobacteriaceae, acinetobacter spp., or p. aeruginosa is detected, as these organisms have all been reported to potentially harbor esbls. the pn panel may provide actionable information on antimicrobial susceptibility for some key organisms. however, appropriate antimicrobial therapy for many targets, particularly in areas where resistance is common, may require follow-up culture and susceptibility testing. this is especially true for organisms with mutation-based resistance, such as s. pneumoniae and p. aeruginosa. implementation of these panels for routine clinical testing still requires additional culture or appropriate follow-up by the performing laboratories to ensure thorough evaluation of ast phenotypes. routine detection of viral analytes and atypical bacteria in upper respiratory tract specimens has been demonstrated on previous biofire respiratory panels. this panel demonstrates performance attributes similar to those of the existing panels ( , ) . a notable difference with this panel is the combined identification of viral subtypes that are reported distinctly in other molecular diagnostic tests (e.g., "coronavirus" as a whole, rather than specific identification of hku , oc , etc., or "influenza a virus" with no additional subtype information). while some of these data may be useful for epidemiological purposes, they should not influence treatment and patient care. the pn panel should have similar if not expanded clinical utility in these populations, facilitating faster access to appropriate treatment and improved clinical outcomes ( , ) . the panel also exceeds the utility of previous respiratory panels with the inclusion of legionella pneumophila and the ability to detect a variety of serogroups ( ) . the pn panel provides a method with improved sensitivity for the diagnosis of legionnaires' disease, which is estimated to account for % to % of cap. the current standard is a urine antigen test, which has a sensitivity of only % and is limited to detection of serogroup , while studies have shown that the use of pcr has improved sensitivity over the current gold standard ( ) . the results for mers-cov are masked in the pn panel product that is fda cleared and available in the united states. this analyte is reported in the biofire pnplus panel, which is sold outside the united states and has also been cleared by the fda with a modified intended use to specifically aid in the differential diagnosis of mers-cov infections only in cases meeting mers-cov clinical and/or epidemiological criteria. the pn panel is intended for the use of both sputum and bal fluid. concurrent bacterial cultures have shown high rates of correlation between sputum and bal specimens ( ) . while viral detection is traditionally done with nasopharyngeal samples, studies comparing use of nasopharyngeal swabs and bal specimens using the biofire filmarray respiratory (rp) panel (an off-label use of the product) have displayed high levels of correlation, with bal specimens generally having a higher diagnostic yield ( , ) . a weakness of this study was that a majority of the specimens enrolled were from hospitalized patients, but this likely reflects the severity of illness in this population and adherence to guidelines suggesting that diagnostic testing is not warranted in ambulatory patients. the data from this study indicate that specimens collected from hospitalized patients and those in outpatient settings had similar incidences of most analytes. the use of a panel that provides sensitive and specific detection of respiratory tract pathogens has been shown to improve patient outcomes and is a recommended tool for antimicrobial stewardship initiatives ( ) ( ) ( ) . the pn panel expands on these existing technologies to provide an easy-to-use, rapid sample-to-answer platform that can detect viral entities and atypical bacteria known to cause pneumonia, in addition to providing a semiquantitative result for commonly encountered bacterial analytes. an earlier study using an ruo version of the pn panel on bal from patients suspected of having ventilator-associated pneumonia concluded that the panel would provide data that could guide appropriate management in this patient population ( ) . the occurrence and impact of viral and bacterial coinfections in pneumonia are not well characterized, but recent studies have shown that coinfection is not unusual in community-acquired pneumonia in adults and was responsible for higher morbidity and mortality ( ) . therefore, it is anticipated that the pn panel could significantly affect the management of patients with coinfections. current algorithms for the diagnosis of pneumonia can include multiple methods; molecular methods are most common for viral agents and many atypical bacteria, and culture remains the gold standard for the diagnosis of bacterial pneumonia. culture suffers from lower sensitivity than molecular methods, in addition to variable methods of interpretation and reporting among and within an institution. culture can also take an average of to h for actionable results to become available. implementation of the pn panel will require consideration of appropriate test utilization in individual patient populations, but it has the potential to be a powerful decision-making tool for patient management. this panel could be utilized for rapid de-escalation or initiation of antibiotics and promoting improved patient care outcomes. further studies are needed to evaluate the clinical impact of this panel and the significance of molecular detection in the absence of culture confirmation. some of the data from this trial have been examined to determine the potential impact on patient care ( ) . estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in countries: a systematic analysis for the global burden of disease study adults hospitalized with pneumonia in the united states: incidence, epidemiology, and mortality deaths: final data for clinical and economic outcomes of hospital acquired pneumonia in intra-abdominal surgery patients a -year prospective study of a urinary antigen-detection test for streptococcus pneumoniae in community-acquired pneumonia: utility and clinical impact on the reported etiology contribution of a rapid influenza diagnostic test to manage hospitalized patients with suspected influenza procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections bacterial and viral co-infections complicating severe influenza: incidence and impact among u.s. patients, - procalcitonin levels in acute respiratory infection management of community-acquired pneumonia in infants and children older than months filmarray pneumonia panel instruction booklet rfit-asy- / practical comparison of the biofire filmarray pneumonia panel to routine diagnostic methods and potential impact on antimicrobial stewardship in adult hospitalized patients with lower respiratory tract infections two-sided confidence intervals for the single proportion: comparison of seven methods detections of mrsa with the biofire filmarray pneumonia panel plus in bal and sputum, poster p can an etiologic agent be identified in adults who are hospitalized for community-acquired pneumonia: results of a one-year study infectious diseases society of america/american thoracic society consensus guidelines on the management of communityacquired pneumonia in adults molecular diagnosis of sepsis: new aspects and recent developments multicenter evaluation of the biofire filmarray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis comparison of three different methods for detection of shiga toxin-producing escherichia coli in a tertiary pediatric care center development and laboratory evaluation of a real-time pcr assay for detecting viruses and bacteria of relevance for community-acquired pneumonia quantitative detection of streptococcus pneumoniae from sputum samples with real-time quantitative polymerase chain reaction for etiologic diagnosis of community-acquired pneumonia ctx-m enzymes: origin and diffusion first report of the emergence of ctx-m-type extended-spectrum betalactamases (esbls) as the predominant esbl isolated in a u.s. health care system ctx-m: changing the face of esbls in europe multicenter evaluation of biofire filmarray respiratory panel for detection of viruses and bacteria in nasopharyngeal swab samples evaluation and implementation of filmarray version . for improved detection of adenovirus respiratory tract infection impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients impact of multiplex polymerase chain reaction testing for respiratory pathogens on healthcare resource utilization for pediatric inpatients diagnostic accuracy of pcr alone and compared to urinary antigen testing for detection of legionella spp.: a systematic review correlation between sputum and bronchoalveolar lavage fluid cultures comparison of respiratory pathogen detection in upper versus lower respiratory tract samples using the biofire filmarray respiratory panel in the immunocompromised host filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples implementing an antibiotic stewardship program: guidelines by the infectious diseases society of america and the society for healthcare epidemiology of america impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections the rapid diagnosis of viral respiratory tract infections and its impact on antimicrobial stewardship programs evaluation of the biofire pneumonia panel in icu patients with suspected ventilator-associated pneumonia impact of bacterial and viral coinfection in community-acquired pneumonia in adults we offer our sincerest thanks to the dedicated laboratory professionals who made this work possible.this study was designed and funded by biofire diagnostics. filmarray testing was performed at the clinical trial sites, comparator culture was performed at mriglobal, and additional discrepancy testing was performed at biofire diagnostics. b. buchan has received speaker's honoraria from biofire; a. harrington has received speaker's honoraria and research funding and is a member of the biofire scientific advisory board; j.d.b. is a consultant to biofire; c. rindlisbacher, m. buccambuso, a. clark, m. rogatcheva, c. graue, and k. m. bourzac are employed by biofire. key: cord- - nfklun authors: eroglu‐ertugrul, nesibe gevher; yalcin, ebru; oguz, berna; ocal, turgay; kuskonmaz, baris; emiralioglu, nagehan; dogru‐ersoz, deniz; ozcelik, ugur; tezcan, ilhan; kiper, nural title: the value of flexible bronchoscopy in pulmonary infections of immunosuppressed children date: - - journal: clin respir j doi: . /crj. sha: doc_id: cord_uid: nfklun objectives: to demonstrate the value of flexible bronchoscopy (fb) and bronchoalveolar lavage (bal) when determining causes of lung infection in immunocompromised children; to investigate differences in causes and radiological features of lung infections following bone marrow transplantation (bmt) compared to other immunosuppressive conditions; to evaluate the reliability of radiological findings when predicting the pathogen. methods: we retrospectively evaluated immunosuppressed children who underwent fb and bal because pulmonary complications between january and may at the hacettepe university hospital pediatric pulmonology unit. two groups, group i (n = ) and group ii (n = ), consisted of patients who had primary or secondary immunodeficiency and those who were immunosuppressed because bmt, respectively. radiological findings before fb and macroscopic and microscopic findings of the procedure were evaluated. results: fb and bal were diagnostic in / patients ( . %) and the antimicrobial treatment changed for / patients ( . %). the most common pathogen was bacteria (streptococcus pneumoniae was the leading one). bacteria were more frequent in group i than group ii (p = . ). no significant difference in radiological findings between groups i and ii was found. considering all patients, a significant association was detected between viral pathogens and radiologically interstitial infiltration and a ground‐glass appearance (p = . ). however, no significant association was detected between bacterial and fungal pathogens and the radiological findings. conclusion: in immunosuppressed patients, fb and bal should be evaluated early for clarifying the causative agents. then, appropriate treatments can be utilised and the side effects and high cost of unnecessary treatment may be mitigated. eroglu-ertugrul et al. pulmonary problems constitute a major cause of mortality and morbidity among patients with immunosuppression. [ ] [ ] [ ] currently, flexible bronchoscopy (fb) is included as a routine diagnostic tool for immunosuppressed patients when respiratory findings (clinical or radiological) are present. , a special group of individuals with immunodeficiency consists of those who have received bone marrow transplantation (bmt) because diseases such as malignancies, hematological diseases, primary immune deficiencies and other hereditary disorders. pulmonary infiltration develops in % of patients who receive chemotherapy for malignancy and this significantly affects mortality. lack of early diagnosis and treatment for the aetiology causing the pulmonary problems in these patients adversely affects their prognosis. the interpretation of pulmonary radiological findings in immunosuppressed patients can also be quite difficult. the appearance of pulmonary infiltration also occurs because noninfectious causes such as the development of graft versus host disease (gvhd) following bmt, disease recurrence or secondary malignancy infiltration, toxicity from chemotherapy or radiotherapy. , in addition, an impaired inflammatory response as well as other predominant atypical and/or nonspecific aetiologies can cause an accurate aetiology determination to be difficult. empirical antibiotic treatment is usually initiated as soon as possible in these patients because of high morbidity and mortality rates, and any delay in treatment can adversely affect the immunocompromised patients' prognosis. , many empirical treatment studies have reported that fb and bronchoalveolar lavage (bal) yield diagnostically valuable results. , it has also been reported that microorganisms can be found or other diagnoses can be made through fb or bal in - % of children and adults with immunosuppression. in such patients, the use of fb and bal can provide a definite diagnosis and as a result, the appropriate treatment can be initiated. the purpose of the study was to demonstrate the value of fb and bal in determining the cause of lung infections that develop in immunocompromised children, to investigate differences between the causes and radiological features of lung infections following bmt in comparison to other immunosuppressive conditions and to evaluate the reliability of radiological findings for predicting the causative pathogen. our study retrospectively evaluated the charts of immunosuppressed children whose data were retrieved from patients who underwent fb and bal between january and may . the patients' demographic features, diagnoses, bronchoscopy indications and the entry route of the bronchoscope, complications because the procedure were all recorded. in addition, preoperative pulmonary radiological findings (direct radiography and/or hrct), the macroscopic findings of the procedure and the results of the microscopic studies of the bal were evaluated. the diagnostic yield of fb and its impact on the management were evaluated. because the patients experiencing severe respiratory distress and/or thrombocytopenia, we applied fb at the appropriate time, but during the procedure all patients were taking broad-spectrum antibiotics and/or antifungal therapy (all patients were taking multiple antibacterial agents, patients were taking antifungals, patients were taking antivirals). the procedure was conducted with an olympus® flexible bronchoscope that included . mm, . mm, . mm and . mm external diameter options. an intubation cannula was used for entry with patients who had already been connected to mechanical ventilators and for the remaining patients, either laryngeal masks (n = ) or the nasal cavity route were utilised. bal was performed from the focal area of the radiological pathology when present and the right lung middle lobe when widespread involvement was present. this study was approved by the institutional review board of the hacettepe university faculty of medicine. we evaluated the patients' radiological findings (chest x-ray and hrct) prior to the bronchoscopy, which were categorised as atelectasis, consolidation, ground-glass appearance, interstitial infiltration, nodular infiltration, bronchiectasis, increased aeration/air trapping, mosaic pattern, chronic changes, lymphadenopathy, mass and airway anomaly. a majority of the patients exhibited more than one finding and in these patients, the most significant/difference-making finding was determined for each patient. we performed cytological evaluations and microbiological studies of the bal fluid. microbiologically, the bal fluid was evaluated for aerobic bacteria, fungus and tuberculosis (tb). furthermore, we utilised pcr to evaluate the presence of respiratory viruses (bocavirus, coronavirus oc / hku , enterovirus, human rhinovirus, influenza a, influenza b, parainfluenza , parainfluenza , parainfluenza , parainfluenza , rsv a, rsv b, metapneumovirus and coronavirus /nl ). any observed pulmonary infection because cmv was defined as a viral load > copies in the bal fluid. immunofluorescence staining methods were utilised (indirect fluorescent antibody (ifa)) to detect pjp. we performed the statistical analyses with the ibm spss for the windows version . software package. the descriptive statistics were calculated using the data obtained from the analyses (percentage, frequency, mean ± standard deviation and minimum-maximum). in addition, we carried out chisquare tests to compare the quantitative variables (pearson's chi-square and fisher's exact chi-square tests) and a significance level of p < . was accepted. we reviewed the hospital records and charts of immunosuppressed patients ( female and male) who were evaluated with fb and bal. the age range was months to . years (mean, . ± . y; median . y). the patients were divided into two groups: group i (n = , %) included patients who had exhibited primary or secondary immunodeficiency and group ii (n = , %) included patients who were immunosuppressed after bmt. to provide more detail, the diagnoses and characteristics of the patients in groups i and ii are presented in table . • in radiological evaluations prior to fb, consolidation in / patients, atelectasis in / patients, diffuse/ local nodular infiltration in / patients, interstitial infiltration in / patients, a ground-glass appearance in / patients and bronchiectasis in / patients were detected. normal findings were present in the remaining / patients. some patients had more than one radiological finding; there were / patients with only one finding, / patients with any two of them and / patients with three of them (detailed radiological results are given in the supplementary table). • the macroscopic evaluation of fb provided findings consistent with infection in patients ( %), tracheomalacia was observed in patients, bronchomalacia in patients, airway anomaly in patients, mucosal hyperemia or hemorrhage in patients and normal findings were present in patients. • in the microscopic evaluations of bal, a microbiological agent was detected in patients ( . %) ( difference was determined for microbiological agents in this group, viral agents were the most common. the microbiological agents detected in the bal are provided in table . . comparisons of microbiological and radiological findings of patients in group i and group ii (table ) • bacterial pathogens were more prevalent in group i than in group ii (p = . ), but the results revealed no difference between two groups in regard to the presence of fungi, tb, viruses and pjp. • additionally, no difference was detected between the groups based on the radiological findings (atelectasis, consolidation, nodular infiltration, interstitial infiltration, ground glass and bronchiectasis). . one of our aims was to determine the role of radiological findings in predicting the causative pathogen group. when all of the patients were considered together, a significant association was determined between the presence of viral pathogens (including cmv) and the radiological findings of interstitial infiltration and/or a ground-glass appearance (p = . ). however, no significant association existed between the radiological findings and the presence of bacterial or fungal pathogens. in the bal sample, of patients ( . %) who were able to be tested for viral agents by a pcr method were found to be positive for viral agents. of these patients, ( . %) had interstitial infiltration and a ground-glass appearance. likewise, of patients ( . %) with a negative result on the viral pcr study did not have interstitial infiltration and/or a groundglass appearance. the predictive values of the aetiologic agent based on the radiological findings are provided in table . . an evaluation of all diagnostic methods related to fb (macroscopic pathologic findings and demonstration of a microbial agent in bal) revealed a diagnostic finding in of patients ( . %). the antimicrobial treatment changed for / patients ( . %); it was escalated based on the identified pathogenic agent in of these patients ( . %): • ganciclovir was given to patients because of pulmonary cmv infection, • anti-tb treatment was started in patients, • a new antibacterial was added for patients • a new antifungal was added for patients however, empirical antibacterial treatment was narrowed in patients because the pathogenic agent not being clearly identified and the competence of the other treatments already in use. treatment changes because the bal microbiology results are provided in table . . complications because the fb were procedure presented in of the patients ( . %), including mild and temporary hypoxia in patients, hemorrhage in patient and temporary bradycardia in other patient that resolved when the procedure was discontinued and did not reoccur during follow-up. no complications from the fb procedure resulted in permanent morbidity and/ or mortality. our results revealed that even though all of the patients received broad-spectrum antibiotics and/or antifungal therapy throughout the procedure, the fb and bal examinations provided significant data in / patients ( . %) that was haemophilus influenzae haemophilus haemolyticus - enterococcus faecium - pneumocystis jiroveci - aspergillus species cytomegalovirus and children with immunodeficiency. in these studies, treatment changes occurred in %, % and % of patients whose bal samples were positive for microbiological studies, respectively. likewise, treatment changes were also reported in . %, % and %, respectively, of the patients whose bal samples were negative for pathogenic agents. in our study, among all patients whose treatment was changed, / patients ( . %) had their treatment increased, whereas only / patients ( . %) had their treatment modified to narrow their current treatment (table ). in the overwhelming majority of the patients in group i, the rate of therapy escalation (n = , . %) was more prominent, especially the addition of antibiotics ( / vs / patients), because bacterial pathogens were more prevalent in group i than in group ii (p = . ). bacterial diversity was high in bal results and the antimicrobial agents at the time of fb did not cover most pathogens. other recent group i (n = ) group ii (n = ) p value reports investigating treatment changes also showed higher escalation as compared to de-escalation and continuation of treatment with nontargeted agents following negative bals ( . %) , although these differences were not as high as the rates we found. we explain this slight discrepancy because in our immunosuppressed population, after observable positive bal results, adding therapy seems reasonable, but the cessation of antimicrobial agents is more difficult in this critical population. in addition, we included patients who changed from prophylaxis to a treatment dose for some antibiotics (such as trimethoprim-sulfamethoxazole and ampicillin-sulbactam) and lengthening the duration of current treatment as a treatment change and this would count as an additive effect to our escalation rate. additionally, these findings point out the overuse of antibiotics and the tendency to continue their use, even in cases without any objective evidence supporting their use. our results also provided evidence that bal evaluation proved to be a very valuable approach for detecting a variety of aetiologic agents such as cmv, tb and resistant microorganisms. our research also investigated whether there were differences in aetiological agents and radiologic features according to which pulmonary infections had developed following bmt as well as in other immunodeficiency states. we determined that in the bmt group there were fewer bacterial agents than in the group with other immunodeficiencies, but no difference appeared for other aetiological agents. these results may be related to the protective effect of prophylactic antibiotics that had been administered since the beginning of the bmt process as well as the preparation regimens that cause t-cell depletion in order to prevent the occurrence of graft versus host disease, both of which may play a critical role in the increased frequency of the presence of viral and opportunistic agents in bmt patients. our results also revealed that bacterial agents, in particular, s. pneumoniae, which is recognised as the most common cause of community-acquired pneumonia in patients with immunodeficiency other than bmt, are also extremely important agents to be reviewed. another aim of our investigation was to evaluate the sensitivity of hrct for predicting causative pathogens before fb and bal had been conducted. in general, practice, bacterial, viral and fungal infections are suspected, respectively, as the causative agents in the presence of consolidation and atelectasis, infiltration and a ground-glass appearance and a nodular appearance. the empirical treatment is determined and initiated accordingly. our study demonstrated that only interstitial infiltration and a ground-glass appearance on ct were significantly related to cmv and other viral agents in bal, but their success in showing the bacteria and fungi detected in bal was low. importantly, the lack of an expected pathogen because immune dysregulation may have also been the reason for this finding. studies in recent years on the role of radiological findings in detecting infectious agents have only specified opacities, nodules and a ground-glass appearance as being associated with a pathogenic factor. in addition, rizik et al. evaluated children with immunodeficiency and determined that although bal results led to a treatment change in % of the cases, the type of radiological findings (focal or lobar vs diffuse, age > vs < ) was not ultimately associated with any treatment change. therefore, our study appears to be the first to evaluate the reliability of radiological findings in predicting the aetiologic agent in children with immunodeficiency as well as the association of viral aetiologies with radiological findings. according to the data from our study, it is recommended that viral investigations be initiated at an early period of treatment as well as to start early antiviral treatment in patients who present with a ground-glass appearance on the hrct. additionally, we recommend that these findings continue to be investigated in studies with larger patient group cohorts. limitations of our current study include its retrospective nature as well as the use of prophylactic antibiotics and antivirals by all patients prior to the study; however, previous research in the past and other recent similar investigations were also conducted under empirical treatment conditions. , , the fact that we could not evaluate the number of colony-forming units that grew in the bal is another major limitation of our study. contamination from the oropharyngeal flora cannot be ruled out, especially in patients with more than one isolated pathogen. however, we have considered these results when managing the therapy. no serious complications because bronchoscopy have been detected in the juvenile age group among immunocompromised patients as found in our study and reported in the previous literature; , however, this is not the case in adult patients with immune deficiency, who exhibited a higher incidence of serious complications. in conclusion, fb and bal should be done in these patients as soon as possible in order to clarify the causative anti-tb addition (n = ) -antifungal addition (n = ) anti-bacterial addition (n = ) narrowing the empirical treatment (n = , . %) n = ( . %) n = ( . %) applications of flexible fiberoptic bronchoscopes in infants and children use of bronchoalveolar lavage in immunocompromised children with pneumonia fiberoptic bronchoscopy and bronchoalveolar lavage for the evaluation of pulmonary disease in children with primary immunodeficiency and cancer. pediatr blood cancer the role of rigid and flexible bronchoscopy in children pediatric bronchoscopy guidelines pulmonary infiltrates in neutropenic patients with acute leukemia during chemotherapy: outcome and prognostic factors prognostic factors of non-hiv immunocompromised patients with pulmonary infiltrates the clinical importance of bronchoalveolar lavage in allogeneic sct patients with pneumonia diagnostic yield of bronchoscopy with bronchoalveolar lavage in febrile patients with hematologic malignancies and pulmonary infiltrates utility of bronchoalveolar lavage in immunocompromised children: diagnostic yield and complications a comparison of bronchoalveolar lavage versus lung biopsy in pediatric recipients after stem cell transplantation bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation bronchoalveolar lavage (bal) in immunocompromised children: years single center experience cytomegalovirus load in bronchoalveolar lavage fluid: a clue to the diagnosis of cytomegalovirus pneumonia? diagnostic yield of bronchoalveolar lavage in immunocompromised children with malignant and non-malignant disorders the utility and safety of flexible bronchoscopy in critically ill acute leukemia patients: a retrospective cohort study bronchoscopy and bronchoalveolar lavage in the diagnosis and management of pulmonary infections in immunocompromised children key: cord- -ig j z authors: couetil, laurent; cardwell, jacqueline m.; leguillette, renaud; mazan, melissa; richard, eric; bienzle, dorothee; bullone, michela; gerber, vinzenz; ivester, kathleen; lavoie, jean-pierre; martin, james; moran, gabriel; niedźwiedź, artur; pusterla, nicola; swiderski, cyprianna title: equine asthma: current understanding and future directions date: - - journal: front vet sci doi: . /fvets. . sha: doc_id: cord_uid: ig j z the havemeyer workshop brought together researchers and clinicians to discuss the latest information on equine asthma and provide future research directions. current clinical and molecular asthma phenotypes and endotypes in humans were discussed and compared to asthma phenotypes in horses. the role of infectious and non-infectious causes of equine asthma, genetic factors and proposed disease pathophysiology were reviewed. diagnostic limitations were evident by the limited number of tests and biomarkers available to field practitioners. the participants emphasized the need for more accessible, standardized diagnostics that would help identify specific phenotypes and endotypes in order to create more targeted treatments or management strategies. one important outcome of the workshop was the creation of the equine asthma group that will facilitate communication between veterinary practice and research communities through published and easily accessible guidelines and foster research collaboration. the effort to clarify the phenotype and terminology used to characterize horses with chronic inflammatory airway disease started in with a workshop in east lansing, michigan ( ) . several workshops were subsequently held with similar goals in mind with the latest hosted in cabourg, france in ( ) . in the last few years, the terminology has further evolved with the term equine asthma (ea) now being recommended to describe horses with chronic respiratory signs ranging in severity from mild to severe that were previously referred as inflammatory airway disease and recurrent airway obstruction, respectively ( ) . although strong evidence supports the role of exposure to environmental dust in the pathophysiology of both mild and severe ea, the potential role of infectious agents (bacterial and viral) has not been clearly established. the goal of the havemeyer workshop on equine asthma was to bring together researchers and clinicians from different disciplines who are actively investigating airway inflammation to discuss the latest information on this topic and provide some comparative perspective from human asthma. the workshop was designed to facilitate productive discussions that would inform potential future revisions of the american college of veterinary internal medicine (acvim) consensus statement on mild-moderate ea ( ) and provide future research directions. the present report follows the format of the workshop. the manuscript is organized thematically starting with the recent advancements in the understanding of the classification and diagnosis of human and equine asthma. the second part is centered on the etiology and pathophysiology of ea. the third and final section of the manuscript summarizes the extensive discussions conducted during the workshop with the goal of prioritizing future directions of ea research. clinical asthma phenotypes have been recognized for many decades but were collapsed into a unified hypothesis of asthma as an allergic disease in when age adjusted levels of immunoglobulin e were associated with asthma. it has taken more than years to consider the heterogeneity of asthma again with an emerging emphasis on endotypes, an intrinsically more interesting approach to understanding asthma pathobiology ( ) . the term "endotype" is used to describe a subtype of disease defined by a molecular mechanism, genetic variation or by treatment response ( , ) . cluster analyses of asthma cohorts have revealed groups with different ages of onset, lung function, concordance or lack thereof between measures of airway inflammation by sputum analysis and symptoms. a recent review of asthma by a panel of experts has focused on the need to recognize asthma in its diverse forms and to identify treatable traits. this extensive review has highlighted areas for future attention ( ) . the application of the analysis of gene expression to airway epithelial cells and sputum cells from well-characterized groups of asthmatics has led to the appreciation of asthma associated with t helper cytokines and non-t asthma ( ) . the former is the more allergic subset with higher ige and peripheral and sputum eosinophilia. non-t asthma has fewer of these features and is less responsive to inhaled corticosteroids. t cells that express interleukin- have been linked to severe neutrophilic asthma. these so-called th cells have been shown in animal models to be associated with steroid-unresponsiveness. the th cytokine interferon-γ likewise has been found to be expressed in the airways of severe asthmatics. in recent years there has emerged another lymphoid cell that participates in host responses to mucosal injury. these innate lymphoid cells are lineage negative, lacking the usual lymphocyte surface markers ( ) . they express similar panels of cytokines to the t helper subsets and are labeled innate lymphoid cell (ilc) , , and . they are rapidly activated by epithelial signals such as thymic stromal lymphopoietin (tslp), interleukins and , molecules termed alarmins. the secretion of il- and il- by ilc may lead to a pattern of inflammation previously interpreted as th . innate lymphoid cells are less steroid sensitive. additionally, alarmins prime cells such as dendritic cells and therefore may have a role in adaptive immunity as well as innate immune responses. the synthesis of amphiregulin, an epidermal growth factor receptor ligand, by ilc s but also th cells, is postulated to promote mucosal integrity. one could anticipate that viral infection of epithelial cells or damage by irritants giving rise to inflammation mediated by ilcs. however, their roles have yet to be fully explored. transcriptomic analysis of sputum has revealed three patterns of inflammation and gene signatures consistent with both th and ilc driven inflammation and oxidative stress ( ) . the descriptions of molecular mechanisms of inflammation may still be considered as a deeper form of phenotyping. however, the application of novel biologics to treat asthma is now implicating certain pathways in disease and therefore is providing us with true disease endotypes. most of the progress in the identification of treatable traits has related to the t phenotype. biologics targeting ige (omalizumab), il- and therefore, the eosinophil (mepolizumab, benrazilumab, rezlizumab) and the t cytokines (dupilumab) have all demonstrated efficacy in reducing exacerbations of asthma. recent results of studies targeting the alarmin tslp and therefore both t high and low asthma have confirmed efficacy against acute attacks of asthma. oxidative stress in asthma has not been specifically addressed. a problematic form of asthma is that associated with airway remodeling and fixed airway obstruction. the association with mucus plugging and eosinophilic inflammation has been recently identified as a potential factor in long term impaired airway function ( ) . severe equine asthma is typically a neutrophilic form of asthma although expression of t cytokines has been described ( ) . there is also evidence that il- is expressed in equine asthma and its effects on neutrophil survival are steroidinsensitive ( , ) . although neutrophilic human asthma is less steroid-sensitive than the eosinophilic phenotype, severe equine asthma is responsive to steroid treatment despite the presence of neutrophilic inflammation. severe equine asthma shares the structural remodeling of the airways with human asthma and a part of the remodeling change is reversible with steroid treatment as well as withdrawal from the inciting stimulus ( ) . studies of airway remodeling in human asthma with treatment have not addressed key components of remodeling such as increased airway smooth muscle mass. revised in and discussed the use of ea to describe these conditions ( ) . the revised consensus recognized that asthmatic horses of all severities have common clinical presentations (such as chronic cough, excess mucus, poor performance) but also a wide heterogeneity in terms of triggering factors, severity, and pathologic characteristics. a phenotype is the observable physical properties of an organism, including measurable laboratory findings, which is the result of the expression of the genes in response to the environment ( ) . identifying distinct phenotypes is of interest if they facilitate the diagnosis, the prognosis or allow the implementation of targeted therapy. while currently loosely defined, the ea phenotypes discussed in the consensus statements were based on clinical presentation (severe vs. mild/moderate), triggering factors (barn/hay or pasture), endoscopy findings (mucus) and bronchoalveolar cytology. from a clinical standpoint, further dividing ea as distinct "mild" and "moderate" phenotypes may promote recognition that asthma is an underdiagnosed cause of exercise intolerance in high performance horses. horses with a cough or increased respiratory rate at rest or following exercise will commonly undergo further diagnostic procedures to confirm asthma, or "anti-asthma" treatments will be implemented. however, this is generally not the case when no clinical signs suggestive of an airway disease are present. the term "mild ea" could describe the condition affecting these horses, while "moderate ea" would be used when clinical signs of airway disease (such as cough) are present, but without the periods of labored breathing at rest seen in "severe ea" ( ) . the inflammatory airway cell phenotypes (neutrophils, mast cells, eosinophils) were recognized in the and consensus statements ( , ) . future phenotypes may include the age (early or late) of appearance of clinical signs, or specific remodeling features affecting the airways, if these new features are shown to facilitate prognostication or the implementation of specific therapy. the future development of new portable and sensitive devices for measuring the lung function of horses (forced oscillation or flow interruption techniques), or the discovery of blood biomarkers for ea would help not only to facilitate the diagnosis of mild and moderate forms of ea in clinical practice, but also to possibly identify new phenotypes for these conditions. to date, different inflammatory pathways have been proposed as contributing to ea, which may eventually lead to novel therapies ( ) . the discrepancies between results of the different studies may be an indication of different endotypes in ea, although future studies on large cohorts of horses from multiple sites would be required before specific endotypes can be recognized. multicenter tissue banking could facilitate these studies. in summary, the acvim consensus statement recognized the currently known distinctive features of ea. further defining "mild" and "moderate" ea based on the presence or absence of easily identified clinical signs may promote the investigation of the subclinical (mild) phenotype. the identification of novel phenotypes and endotypes may lead to "precision medicine" where treatments most likely to help equine patients would be selected. this approach is now implemented in humans and may eventually be applicable to horses if supported by scientific research. severe equine pasture asthma (epa) is characterized by episodes of reversible airway obstruction in horses grazing pasture during the summer in hot humid climates ( ) . affected horses demonstrate neutrophilic airway inflammation, airway hyperresponsiveness extending throughout the season of remission, and airway remodeling ( , ) . the author's experience is restricted to epa as first described in horses residing in louisiana, and diagnosed in states with subtropical climates (mississippi, alabama, and florida) ( ) . veterinarians in regions of adjoining states and distant states (oregon) describe similar signs in horses grazing pastures during hot humid conditions. epa is described in the united kingdom where it differs in its association with hot dry weather or exposure to dust from harvest/burning of crops ( ) . epa demonstrates adult onset ( ± years; range - years) without sex predilection ( ) . asthma exacerbations generally begin in summer (july), persisting until temperature and humidity decrease (october/november) ( ) . fewer horses experience asthma in the spring. a history of prior seasonal cough and/or exercise intolerance may be identified. improvement within hours to days of isolation from pasture particulates in a stall environment is a key diagnostic feature of epa in the southeastern usa ( ) ; some severe cases necessitate isolation in a climate climate-controlled environment. in the author's experience, without adequate environmental management, disease severity is progressive and responsiveness to parenteral corticosteroids decreases. though specific agent(s) that elicit epa exacerbation are not identified, the response to stall housing implicates seasonal pasture-associated particulates. costa et al. reported increases in grass but not tree pollens were significantly associated with epa exacerbation using a pollen station ∼ miles from affected horses ( ) . in this regard, intact pollen is too large to reach the respirable zone of humans in order to elicit asthma, but moist conditions that are associated with epa exacerbations can shatter pollen and disseminate respirable particles ( ) . grass pollen sensitization is classically associated with th responses, ige-mediated hypersensitivity, and eosinophilic inflammatory infiltrates. however, chronic exposure to th sensitizing antigens and to complex antigen combinations that include th sensitizing antigens each generate th responses accompanied by neutrophilic airway inflammation that typifies epa ( , ) . subtopical grasses differ substantially from grasses in temperate and continental climates ( ) . pollen from subtropical grass subfamilies is important to rhinitis and human asthma in subtropical zones of australia, asia, india, africa, and america. pollination seasons for bahia and bermuda grass (spring through september/october) align to the season of epa exacerbation ( , ) . the pollen season for johnson grass is temperature dependent, flowering from may to july, with higher temperatures moving flowering later into autumm ( ) . a role for fungal triggering in epa exacerbation is suggested by the near identical clinical picture presented by horses with epa and barn dust-associated severe asthma, wherein a role for fungal triggering is substantiated in the latter ( ) . chronic neutrophilic airway inflammation characterizing both forms of severe equine asthma also aligns to th -mediated neutrophilic inflammation in fungal asthma models ( ) . of the more than species of fungi that exist in biotropic relationships with bermuda, bahia, and johnson grasses, curvularia, helminthosporium, alternaria, puccinia, epicoccum, and fusarium are implicated in eliciting human asthma ( ) . costa et al. identified fungal spores of the genus nigrospora, and curvularia, as well as basidiospores, as temporally associated with exacerbations of pasture asthma ( ) . these findings are in agreement with reported correlations between epa exacerbation and high dew point temperature ( ) . specifically, nigrospora conidia and basidiospore release increase with increasing relative humidity, resulting in a peak in spore counts during the early morning and aligning to the association of epa exacerbations with increased dew point temperature ( , ) . in contrast, conidia of cladosporium, alternaria, epicoccum, and dreschlera spp. are released during warm, dry, windy conditions, while precipitation is required for release of many ascospores. in this way, humidity influences fungi of relevance to asthma in different locales which could influence associations of pasture asthma in the uk with hot dry conditions, rather than hot humid conditions precipitating pasture asthma in the southeastern us. as a chronic and progressive disease of undetermined etiology, epa is most effectively managed by segregation from inciting grass pastures during warm seasons. the necessity to segregate horses from pasture, particularly at a time when they are typically extensively ridden and grazed, presents a conundrum that is ultimately detrimental for most affected horses. accordingly, there is a critical need to identify the agents that trigger epa in order to improve disease management. both veterinary practitioners and researchers muse about the diagnostic armamentarium available to physicians-if only we had the chest ct, the advanced lung function testing, the biomarkers-then we would be able to have a better diagnosis. a quick search of the literature, however, shows us that our counterparts face many of the same diagnostic dilemmas that we do, albeit often with higher bills! while pulmonologists have drawn up multiple guidelines to help in the diagnosis of asthma in humans with its multiple phenotypes and endotypes, physiciandiagnosed asthma criteria often fail to be consistent with the official guidelines rendering the results of large epidemiologic studies or clinical trials fraught with the perils of resting findings on nebulous datasets. various forms of spirometry or simple pulmonary function testing are readily available in human medicine, but few non-pulmonologists avail themselves of objective data, and instead rest on reported symptoms such as difficulty breathing on exertion, cough or positive response to bronchodilation ( ) . indeed, the gina toolbox identifies "lack of access to spirometry/bronchoprovocation tests" as a barrier to implementation of gina guidelines in human asthmatics ( ) . moreover, the heterogeneity in published algorithms for diagnosis of asthma-more than in the literature at last count-make even an algorithm-based diagnosis unsure ( ) . thus, the conclusion that symptom-based diagnosis is associated with a significant risk of over-diagnosis has been reached for asthma in humans ( ) . the current push in human medicine to refine both the phenotypes and endotypes for multiple different subtypes of asthmas aims to elucidate the underlying causes and thus treatments that may be very different. we are still searching for the criteria that will help us with this in equine medicine. if there are indeed mechanistically different groups of horses within the categories of mild, moderate, and severe ea that are associated with genetic differences or cellular or molecular biomarkers, then perhaps we will gain better understanding of treatment successes and failures and will be able more logically to choose clinical therapies and predict responses. the difficult case for the clinician and the researcher alike is not the horse with severe ea-because the history and clinical exam alone can often suffice to diagnose, and there is a visible relief in respiratory embarrassment with administration of bronchodilator (although it can take some time in horses with diaphragmatic exhaustion) ( ) . the difficult horse is the one with moderate/severe asthma in remission and the horse with mild-moderate ea. as was recently pointed out, the biggest difference that we note in the clinical diagnosis of horses with mild-moderate ea vs. severe ea is the presence of an increased respiratory effort at rest, which is due to the underlying pathophysiology of bronchoconstriction, increased mucus, and bronchiolar inflammation ( ) . the need, then, is to detect the mildly or subclinically affected horse. as veterinarians, we have at hand history, clinical signs, lung function testing, radiographs, endoscopy, analysis of airway secretions, blood biomarkers and clinical pathology which can be used in a minimum database in order to classify horses into clinically useful categories that have a pathophysiologic basis that can simultaneously allow us to diagnose, treat, and translate clinical cases into field research. a tentative diagnosis of ea in its most severe form can often be made on history alone, with the key component being the recognition of episodes of reversible respiratory embarrassment precipitated by exposure to specific triggers-namely, moldy hay in the northeast of the united states, and pasture allergens and particulates in the south. history in subclinical or mild cases is seldom of such definitive use; this does not mean that it is unimportant. such questions as parentage ( ) , type of feed and how it is fed ( ) , and heat and pollen counts at the time of diagnosis ( ) may be important risk factors for equine asthma. while it has been proposed that coughing and poor performance may serve to define a phenotype of moderate vs. mild ea ( ) , these signs are not sufficiently sensitive ( ) and would misclassify a subset of horses-they alert the clinician that moderate ea is likely, but the absence of these signs does not rule out disease. the connections between ea and viral or bacterial disease are not linear, but it is becoming increasingly clear that the connection exists ( , ) , thus a thorough history should include probing for past infectious respiratory disease. one of the best described questionnaire analysis tools for classification of horses based on history is the hoarsi index ( ), developed as a means of distinguishing among normal, mild-moderate ea or severe ea phenotypes. however, clinical signs and indices are insufficiently sensitive to distinguish horses with mildmoderate ea from normal horses or horses with severe ea in remission ( ) . proposed minimum database for both practitioners in the field and for research: a common history tool should be developed that addresses the main concerns of parentage if known, current and lifetime exposures to particulates and allergens including feeds and feeding practices, barn environment, vaccinations, travel history, and recent illnesses. multiple scoring systems have been shown to be useful for distinguishing healthy horses from horses with severe ea in exacerbation, but, similar to questionnaire indices, these scoring systems do not help in the more difficult problem of distinguishing horses with mild ea from healthy or severe ea in remission ( ) . indeed, years ago, robinson et al. found that even in horses with historical severe ea, clinical score failed to reflect low-grade airway obstruction, and suggested that without easily used, field-accessible testing equipment, lower airway disease would go underdiagnosed ( ) . recently, the adapted -point scoring system has been shown to be the most useful in discriminating mild from severe cases, but it is unlikely to distinguish normal from subclinical disease ( ) , and the ideass scoring system has recently been described as a useful scoring system for moderate-to-severe equine asthma ( ) . thus, while clinical scoring is essential to a good examination and careful research, and can potentially be useful in measuring response to treatment in the individual, it is insufficient in making the phenotypic distinction between mildly affected horses and healthy horses. proposed minimum database for both practitioners in the field and for research: the -point modified clinical score appears to best stratify horses with obstruction ranging from mild to severe. an application suitable for smart phone use would enhance the adoption of a common scoring tool. in human asthma, the gold standard is the detection of variability in pulmonary function using spirometry or other methods of lung function testing ( ) . unfortunately, lung function testing remains available only to a few specialized centers, as more recently developed portable lung function testing modalities are no longer on the market ( ) . initial reports from the author's laboratory of a simple field test of respiratory resistance using the interrupter technique hold promise for increased use of lung function testing in the future. while the classic esophageal balloon/pneumotachometer method is effective in demonstrating increased maximal pleural pressure and allows for calculation of pulmonary resistance and elastance as well as dynamic compliance in severe ea, it is not sufficient for demonstrating abnormal function in mildly affected horses in which baseline lung function is rarely abnormal and histamine or other bronchoprovocation or bronchodilation must be used in order in order to detect low-grade obstruction ( ) . unfortunately, in some studies, even histamine bronchoprovocation has not been sufficient to distinguish between normal horses and horses with mild asthma ( ) , and a lack of concordance between histamine bronchoprovocation and bronchoalveolar lavage (bal) cytology has been noted in several studies ( , ) . while lung function testing and histamine bronchoprovocation have shown moderate to strong correlations with bal cytology in some studies ( , , ) , others have not ( , ) . methods of performing histamine bronchoprovocation are equally important: studies in human asthmatics have shown that it is the total dose of histamine that is most important rather than the duration of exposure. a more precise method of dosing may be important to establish. in human athletes, indirect stimuli, such as cold air, hypertonic solutions such as mannitol, exercise, and amp are all considered more accurate and useful in predicting asthma than are direct stimuli such as methacholine or histamine; this is an area that requires exploration in equine pulmonology. while hay challenge is useful for research in severe ea, it is inappropriate in a clinical case, especially in a horse that is expected to do athletic work ( ) . in moderate to severe ea, variability in airflow should be demonstrated not through bronchoprovocation but through bronchodilation using either systemic (buscopan tm ) or inhaled (albuterol, ipratropium bromide) drugs to assess reversibility; it is possible that a h period of bronchodilation is necessary for maximum effect in horses with diaphragmatic fatigue ( ) . proposed minimum database for both practitioners in the field and for research: in research, lung function should be assessed and airflow variability/changes in airway caliber should be assessed with either bronchoprovocation or bronchodilation. more research is necessary to determine if field assessment of lung function after bronchoprovocation or bronchodilation is sufficient to determine change with the -point scoring system. it is essential that a robust, easily used system for testing lung function in the field be developed. unlike in human pulmonology, examination of airway secretions is a primary method of diagnosis in ea, be it mild, moderate or severe. although a standard volume of between and ml of saline using a m long endoscope or m bal tube is recommended ( ), this practice is not always followed, and cytology should be assessed keeping in mind that the amount of fluid infused will affect the cell percentages. the relationship between bal cytology and performance is still not clear. certainly, poor performance has been associated with what have been determined to be abnormal cell types or percentages ( ) . there has been much discussion as to what is normal on bal cytology; it likely depends on a combination of technique, environment and population. even the "stringent" definition proposed by couëtil et al. ( ) of < % neutrophils, % mast cells, % eosinophils, would be considered elevated in some high-performance populations ( , ) . although an earlier study found no evidence of a clear phenotype in mast cell vs. neutrophilic inflammation with respect to pulmonary gas exchange during exercise ( ) , recently, an increase in bal mast cells or neutrophils was shown to negatively affect performance ( ) . the way that cells are counted in bal cytology is also important, especially for rare cells. in our laboratory we count a minimum of cells at x for common cells such as macrophages and lymphocytes or neutrophils in mild ea, whereas for rare cells such as mast cells we count , cells. other techniques, such as using a -field differential for mast cells, are only useful if the cell density is high ( ) . the conundrum of whether to assess airway fluid from both lungs rather than blind sampling, or to pool samples, has also occupied attention from researchers. one group found that, depending on whether the "loose" or "stringent" categorization was used, - % of horses would have been categorized as control vs. mild-moderate ea if only one lung were used ( ) . as it is the rare practitioner who has a bronchoscope in the field, it is unlikely that even pooled samples ( ) , which may be a better representation of overall lung inflammation, will be taken other than in referral centers or practices. the problem is most important for rare cells. more attention will need to be paid in future to morphology and perhaps typing of cells. the existence of neutrophil extracellular traps (netosis) in horses with severe ea presents an additional method to determine response to treatment ( ) , and recently the presence of degenerate neutrophils has been shown to raise suspicion for bacterial infection ( ) . the question of macrophage morphology as an indicator of inflammation is also an area that will profit from further investigation ( ) . recently, as well, the paucigranulocytic phenotype has been described in which horses with clear signs of severe ea have low neutrophil percentages in the bal ( ) . this is thought to be due to mucus plugging of small airways that essentially sequesters neutrophils. although a recent publication showed a rather shocking % of highperforming european horses with mild-moderate ea had fungal elements in the bal ( ) , this remains to be confirmed in other populations. proposed minimum database for both practitioners in the field and for research: for the bal, at least -mls of saline should be used, and there is a preference for counting at least cells to adequately represent rarer cells. for research purposes where rare cells are of interest (e.g., mast cells or eosinophils), sampling of both lungs appears preferable. better categorization of cells through morphological descriptions including apparent neutrophil extracellular traps and notations of fungal or birefringent elements should be done. characterization of mucus on cytology may help to elucidate the paucigranulocytic phenotype. bal in the field will usually be done blindly with a specialty tube. the debate continues to swirl around the utility of tracheal wash vs. bronchoalveolar lavage, with malikides et al. ( ) finding a % disagreement in young racehorses, while derksen et al. ( ) determining that there was no correlation between bal and tw, and others finding no relationship between tracheal neutrophil counts and racing performance ( ); thus, tracheal cytology has been considered inappropriate for diagnosis of mild ea ( ). recently, however, a comparison of tw and bal in horses, along with evidence of mucus and endoscopy, found that only . % of horses would have been classified differently if they had had the other procedure, eventually concluding that there is no gold standard-except for mast cells, which are rare in the trachea, and thus, to be found, demand that a bal be performed ( ) . proposed minimum database for both practitioners in the field and for research: tracheal wash may be most practical for some practitioners in the field and has the added benefit of allowing for bacterial culture. the inability to assess mast cells adequately continues to limit this modality. in research settings, both tracheal aspirate and bal are preferable. many clinical diagnoses are made on the basis of endoscopic visualization of mucus, with strong support from the finding that tracheal mucus quite nicely correlated with racing performance or lack thereof ( ) . the recent consensus statement considers that the demonstration through tracheobronchial endoscopy of mucus grade / in racehorses or / for sport/pleasure horses is sufficient to diagnose mild-moderate ea and in support of this recommendation, rossi et al. ( ) found that visible mucus in the trachea is indeed likely to predict inflammation. there are varying degrees of certainty about mucus in the trachea predicting inflammation ( , , , ) . nonetheless, other studies have shown that mucus is insufficient to parse out mild vs. unaffected cases ( ) . endoscopy has also been shown to be useful in detecting an increase in upper airway abnormalities in horses with mild-moderate ea, with courouce-malblanc et al. ( ) raising the chicken-and-egg question of the relationship between mild-moderate ea and dorsal displacement of the soft palate, and more recently, wysocka and klucinski ( ) found that more horses with mild-moderate ea had dynamic pharyngeal abnormalities. it may be that the answer will rest in whether any of these modalities can help to define a phenotype rather than simply further describing an already understood phenotype. proposed minimum database for both practitioners in the field and for research: upper airway endoscopy should be performed to rule out upper airway cause of obstruction as a primary cause of signs or that might confound lung function testing. assessment of tracheal mucus should be performed. endobronchial biopsies offer an excellent method of sampling larger airways, although deeper layers cannot be accessed. the brass ring-being able to distinguish normal from remission or mild ea-remains elusive, however, as correlates were evident between histopathology and impulse oscillometry and showed a difference between horses in remission at pasture and those that remained stabled and treated with glucocorticoids, but did not show any difference between horses with severe ea in remission and controls ( ) . proposed minimum database for both practitioners in the field and for research: at this time, brushings/biopsies are not considered part of a minimum database. imaging is considered an important ancillary diagnostic in humans, but radiographs have not been shown to be sensitive or specific in horses with ea ( ) . chest ct is currently not feasible in large animals. while endobronchial ultrasound shows promise for the elucidation of airway smooth muscle thickening in severe ea, the ultimate goal of being able to detect low-grade disease in erstwhile healthy horses, or to distinguish normal from severe ea in remission remains elusive ( ) . proposed minimum database for both practitioners in the field and for research: at this time, imaging is not considered part of the minimum database. equine asthma encompasses mild to severe forms of chronic airway inflammation. severe ea affects ∼ - % of horses in countries with northern, cool climate ( , ) . mild-moderate ea affects - % of pleasure horses based on tracheal wash cytology (neutrophils > %) and up to % of racehorses based on bal cytology ( , ) . horses affected with severe ea experience exacerbation of clinical signs when exposed to organic dust originating from hay and bedding, in particular molds present in poor quality hay. as a result, clinical signs tend to be worse during the winter when horses are housed indoors for extended periods of time ( ) . some horses exhibit disease flare-ups while at pasture during summer months (epa) ( ) . these horses improve clinically during winter or after being housed indoor. a small percentage of horses appear to suffer from both classic severe ea and epa. horses with severe asthma tend to be mature (> years) to old animals and a genetic predisposition has been identified in some families ( , ) . the main clinical sign characteristic of severe ea is increased respiratory effort ("dyspnea") that can rapidly improve following bronchodilator administration. although the decrease in respiratory effort following bronchodilator administration can be detected within minutes of drug administration using lung function testing, clinical improvement may not be apparent to clinicians ( ) . acute exacerbation is associated with increased pulmonary artery and right-heart vascular pressures as well as increased pulmonary artery diameter on ultrasound ( ) . blood pressure return to baseline during clinical remission however, cardiac ultrasound abnormalities such as right ventricular wall thickness remained increased ( ) . surprisingly, severe ea is rarely fatal unless complications develop such as cor pulmonale ( ) . affected horses are more likely to be euthanized because owners get discouraged with the expense associated with chronic therapy and maintaining a low-dust environment ( ) . coughing and nasal discharge are non-specific signs of respiratory disease commonly reported in horse with severe ea ( ) . horses with a history of both coughing and mucoid nasal discharge are at increased risk of developing severe ea ( ) . thoracic auscultation may reveal increased breath sounds bilaterally, extended area of auscultation, and abnormal breath sounds (i.e., crackles, wheezes). however, the thick chest wall of horses makes auscultation an insensitive indicator of pulmonary disease, with abnormal findings obtained in < % of horses with severe ea ( ) . strict management changes or medical therapy will results in rapid improvement in clinical signs however, if exposure to triggering factors is not addressed improvement will be short lived or incomplete ( , ) . this form of mild respiratory disease is mainly subclinical with horses showing non-specific signs such as intermittent coughing and poor performance ( ) . however, mild asthma should not be ruled out in horses that do not cough because coughing is reported in only % of horses with mild asthma ( ) . coughing is associated with increased bal neutrophils ( ) . poor performance and reduced willingness to perform are associated with increased tracheal mucus scores in racehorses and show-horses, respectively ( , ) . in racehorses, poor performance has been associated with increased neutrophils and mast cells in bal fluid ( ) . there is an association between nasal discharge and increased tracheal mucus in racehorses ( ) . however, the association between tracheal mucus and bal cytology has not been reported yet. the term "remodeling" defines a process resulting in a tissue that is structurally and architecturally altered compared to its healthy counterpart. in asthma, structural alterations are represented by quantitative or qualitative changes of the bronchial wall components or their surrounding tissues, whilst architectural alterations refer to the skewed relationships among such structures. airway remodeling has been studied only in horses affected by severe ea. an increased expression of metalloproteinases and their tissue inhibitors has been recently reported in a group of horses with mild respiratory signs and bal cytology compatible with mild ea ( ) . however, the possibility that the horses studied were horses with severe ea in remission of the disease was not excluded. almost all airway components undergo remodeling in severe ea, both in peripheral (diameter < mm) and central airways. the airway smooth muscle mass as well as collagen and elastic fiber deposition are increased in the lamina propria of peripheral airways during severe ea remission compared to healthy airways ( , ) . mucostasis, mucus cell hyperplasia, peribronchiolar metaplasia, and interstitial fibrosis are more frequently detected in horses with severe ea in remission compared to controls ( ) . however, histomorphometric techniques revealed no differences in the number of mucus cells per mm of lamina reticularis or in the volume of stored mucosubstance in bronchial epithelial cells ( ) . central airway remodeling during disease remission is less pronounced compared to what is observed peripherally. whether airway submucosal structures are significantly altered during severe ea remission compared to control remain to be established ( , , ) . functionally, severe ea remission is associated with a normal lung function in spite of significant structural alterations of the airways. in these conditions, the respiratory resistance correlates with the amount of collagen within the lamina propria of peripheral airways ( ) , indicating that, in the absence of bronchospasm, peripheral airway stiffness is the major determinant of respiratory resistance in asthmatic horses. the functional implications of peripheral remodeling become more important during disease exacerbations, when most of the changes are further accentuated and the mechanics of breathing are altered ( , ) . there is no doubt that the major determinant of airway obstruction during severe ea exacerbations is smooth muscle contraction and that central airways play a major role ( ) . by definition, the force produced by a muscle is proportional to its cross-sectional area. given the increased smooth muscle mass (and cross-sectional area) during severe ea exacerbations ( ) , asthmatic muscle is "stronger" and able to contract the thickened lamina propria observed in severe ea, further reducing the airway lumen. increased mucus secretions into the airway lumen also contribute to airway occlusion ( ) . these same mechanisms operate in peripheral airways, where the effects on lung function are somewhat blunted by the fact that their overall contribution to pulmonary resistance is low, due to their large cumulative cross-sectional area ( ) . at this level, the more relevant functional effects of remodeling are the loss of lung elasticity and airway-parenchymal tethering. adequate small airway patency is guaranteed by their intimal connection to the lung parenchyma by elastic and connective fibers. when the lung inflates during inspiration, small airways are stretched and passively dilate. remodeling of elastic fibers and of the extracellular matrix within and around the airways and in the alveolar septa alters this mechanism, preventing the smallest airways from remaining open ( ) . the effect is even worse during expiration, when the lungs physiologically recoil and the airway diameter physiologically narrows. with a significantly impaired expiratory airflow, part of the air that reaches the alveoli remains trapped. this leads horses with severe ea in exacerbation to breath at increasing lung volumes [functional residual capacity ( ) ] in the attempt to maintain airway patency, which causes lung hyperinflation and enlarged fields of thoracic auscultation ( ) . anecdotal evidence to date has suggested that, although bal sampling is widely accepted elsewhere as the diagnostic tool of choice for cytological assessment of equine lower airways, tracheal endoscopy and tracheal wash-based diagnostics have remained the mainstay of routine clinical lower airway investigations in british thoroughbred racehorses in training. given the emphasis on bal in research, this would present a considerable challenge to furthering evidence-based respiratory medicine in this important equine population. in a recent study we investigated british racing veterinarians' rationales for current practices, and the challenges they face in relation to diagnosing and managing racehorse airway inflammation ( ) . qualitative data were gathered through semi-structured focus group discussions designed to capture current practices and opinions relating to the diagnosis and treatment of lower airway inflammation, as well as familiarity with and views on the most recent acvim consensus statement ( ), in which the term "mild-moderate equine asthma" was recommended. four british veterinary practices, two primarily serving the flat racing community and two primarily serving the national hunt (jump racing) community, in different geographical regions of england, were purposively selected to participate. focus group discussions were conducted at the practice premises, moderated by one of the authors (tk), an experienced qualitative researcher who is not a veterinarian. discussions were audio-recorded and transcribed verbatim, and transcripts were analyzed using an inductive, thematic analysis. in total, participants contributed to the focus group discussions (number per group ranged from to ). all were veterinarians (experience ranging from recent graduate to senior partner), with the exception of one laboratory team member and one veterinary student, and five were women. discussions lasted between and min. three key themes were developed through analysis of focus group data: (i) an over-arching theme of serving the racing industry within which two further themes (ii) disregarding of the consensus and (iii) the pragmatic clinician were nested. (i) serving the racing industry: this was a key driver of clinical approaches to racehorse respiratory health, which were strongly trainer-influenced in particular. the trainer selects horses for endoscopic respiratory assessment, often because of training and racing schedules rather than any clinical signs, and the approach to investigation and treatment is strongly influenced by trainer expectations. this varies with trainer personality, experience and training methods, as well as stage of the racing season, signalment of the affected animal and general health on the yard, and is in turn driven by commercial pressures of the racing industry. (ii) disregard of the consensus: the unanimous view across all four groups was that the condition defined as mildmoderate ea by current concensus ( ) is largely not seen in british racehorses which, in the participants' considerable collective experience, are affected predominantly with excess endoscopically-visible tracheal mucus largely attributed to bacterial infections. it was also considered unfeasible to fulfill two key aspects of the consensus case definition: waiting for chronicity of clinical signs (> weeks duration), and performing bal sampling. neither of these would be acceptable to trainers, according to participants, and participants themselves were not convinced of the extra value of bal sampling. the consensus statement was therefore seen as having been developed for outsiders, by outsiders without sufficient understanding of culture and practices on british racing yards. (iii) the pragmatic clinician: participants shared a strong professional identity as pragmatic clinicians often required to base clinical decision-making on direct personal or collective experience, rather than on research-based or laboratory evidence. cytological examinations of tracheal wash samples were defended as valuable when interpreted sequentially and combined with knowledge of the history and idiosyncracies of the individual horse and yard. although this approach was generally viewed positively as flexible and individualized, participants did also express some frustration with the sometimes unsatisfactory jigsaw of diagnostic information available to them, particularly in relation to discrepancies between clinical and laboratory findings. our work has highlighted a lack of alignment between clinical practice on british racing yards and international consensus on diagnosing lower airway inflammation, which constitutes a barrier to furthering development of a contextually-relevant evidence-base for this population. equine clinicians elsewhere may find themselves in disagreement with some of the opinions expressed, or practices described, by our study participants. however, these investigations were designed to understand the experiences and rationales of clinicians in the specific context of british racing practice. the strength and consistency of views expressed support the anecdotal evidence that, in this context, tracheal endoscopy and wash sampling are widely regarded as the best available means of providing the non-invasive monitoring of respiratory health expected by trainers and used to inform training-and racing-related decisions. it would be interesting to determine whether similar approaches are being taken elsewhere, particularly in populations of yearling and year old thoroughbred racehorses in training. given the considerable resistance to bal sampling in british racing, development of new tracheal-based or other minimally-invasive diagnostics, including appropriate biomarkers and suitably sensitive, portable lung function tests, would be valuable. furthermore, our participants' views that mild-moderate ea as defined by current consensus is largely not seen in british racehorses suggest that research furthering our understanding of the etiology and pathogenesis of airway inflammation in this equine population is still required. the respiratory system is an interface between the outer environment and the inner body. lower airways have historically been seen as a sterile milieu, thanks to the anatomical configuration, local surface immunity and mucus production and clearance systems ( ) . however, with the development of high sensitivity and high throughput technologies, the microbiota of the respiratory system has been described in healthy subjects in many species, including horses ( , ) . further investigation of the relationship between infectious agents, lower respiratory tract microbiota and the development of mild ea is warranted. we and others have reported descriptive results about the microbiota of horses with mild ea ( , ), but the causality between bacterial flora and the disease is far from being understood. studies on the microbiome use dna extraction followed by high throughput amplification and sequencing of the s amplicon ( ) . the sequences are then filtered and aligned against a taxonomy database to identify and organize operational taxonomic units (otus). descriptive analysis of the phyla, otus and bacterial species are then performed, followed by statistical analysis at the community level (within and between samples; alpha and beta diversity, respectively) and at the individual level (otu diversity analysis). statistical analysis can be used to compare between groups: healthy horses vs. those with mild asthma, upper vs. lower respiratory tract ( ) . the lower airways have a decreased richness (alpha diversity, corresponding to the number and proportion of each bacterial species) when compared to the upper airways in healthy horses ( ) . however, a very large majority of the same otus are present in both the upper and the lower airways, showing an overlap and some continuity in the bacterial population between the two anatomical environments in healthy horses. furthermore, treatment with corticosteroids did not affect the composition of the bacterial flora in the upper airways ( ) . the role of the upper airways microbiota in mild ea is unknown, but two studies did not find any difference in beta diversity of the upper airways between healthy horses and those with mild ea ( , ) . the relationship between bacteria and the lower respiratory tract of the equine host seems to be dynamic. as an example, a change in the environmental respirable particulates has an effect on the lower respiratory tract flora in horses. furthermore, treatment with systemic or nebulized dexamethasone induces some changes in the microbiota of the lower respiratory tract in both healthy and mild asthma horses ( ) . systemic dexamethasone administration decreased the evenness of the flora and increased the abundance of otus. there is an agreement between studies that the lower airways microbiota between healthy and mild ea horses are clearly different ( , ) . interestingly, streptococcus is one of the otus which differed with disease status, and was the otu with the greatest increase in relative abundance in mild ea. the effect of the environment on the composition of the lower airways' microbiota is also a common finding between studies ( , ). however, a study found that treatment with corticosteroids had more effect on the composition of the bacterial flora than changes in the environment ( ) . the microbiome studies are recent in equine medicine and are limited to being descriptive. the challenge for the scientific community will be to answer the causality dilemma of the chicken or the egg regarding the role of the airway microbiota in mild ea. asthma development in humans is most probably caused by the interaction of multiple factors, including genetics, allergen exposure, microbiome and invading pathogens. human rhinovirus, human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus, human enterovirus and human coronavirus are strongly associated with asthma exacerbations ( ) . the association between human rhinovirus-induced wheezing and the development of childhood asthma/wheezing has been confirmed in a recent meta-analysis ( ) . the risk for asthma by age years has been shown to increase (odds ratio . ) if children have been wheezing with rhinovirus during the first years of life ( ) . further, many prospective long-term follow-up studies have shown that human respiratory syncytial virus-induced bronchiolitis is associated with later development of asthma ( ) . however, the pathogenic role of respiratory viruses as triggers for the development and/or exacerbation in asthmatic human patients has not been fully characterized. changes in the immune response to viral infections in genetically predisposed individuals are very likely to be the main factor involved in the association between viral infection and asthma ( ) . the pathogenesis of ea remains incompletely defined. however, similar to human asthma, a multifactorial process is suspected. conditions associated with exercise, feeding and housing practices, location, seasonality, infection of the upper and lower airways and genetic influences have been linked to ea ( , , , ) and bacterial (streptococcus equi subspecies zooepidemicus, actinobacillus spp., pasteurella spp.) etiological agents have been linked to mild to moderate ea ( , ) . it remains to be determined if these agents are triggers for the development of ea or are secondary colonizers of already compromised airways. viral respiratory infections are one of the most common health problems in horses throughout the world ( table ). these infections are often self-limiting and a full recovery can be expected in most horses. young performance horses, such as racing horses, have an increased risk of respiratory viral infections. this relates to age susceptibility, commingling, stress and suboptimal biosecurity protocols ( , , ) . amongst respiratory viruses, only eiv and ervs have an affinity to the lower respiratory tract, leading to airway hyperresponsiveness. clinical signs associated with eiv are usually more severe than those seen with mild to moderate ea. further, no association has been determined between mild to moderate ea and infections with eiv, ehv- and ehv- ( , , ) . this is in sharp contrast to the detection of ervs (erav and erbv), known to cause subclinical or mild clinical disease ( , , ) . in a recent study, horses with mild to moderate ea were significantly more likely to have a positive titer as well as higher log-transformed titers to erav when compared to control horses ( ) . in another study, the detection of erbv by qpcr was significantly associated with coughing in standardbred racehorses in training ( ) . subclinical respiratory viral activity in horses with poor performance has been associated with ehv- and ehv- infection ( , ) . in a recent study, the detection of ehv- by qpcr in nasal secretions was significantly associated with mild to moderate ea ( ) . in another study, the detection of ehv- by qpcr was significantly associated with coughing and excessive tracheal mucus in standardbred racing horses ( ) . these results are in sharp contrast to two recent studies performed on swedish standardbred trotters, which were followed for months via qpcr analysis of nasal secretions and serology ( , ) . despite occurrence of poor performance and subclinical viral activity in the swedish standardbred trotters, the authors were unable to detect associations between ehv- /- and clinical respiratory disease and/or poor performance. these conflicting results reflect the ongoing challenges in establishing causality between mild to moderate ea and gamma herpesviruses, known to be ubiquitous in both healthy and clinically affected horses. in conclusion, associations between specific viruses detected via antigen or antibody detection and clinical signs of mild to moderate ea may suggest that viruses may play a role in triggering or exacerbating asthma. however, because some viruses are ubiquitous both in healthy and clinically affected horses or are often associated with subclinical disease, establishing causality is challenging and in need for further research. a growing body of research demonstrates the link between organic dust exposure and ea. introduction of horses to high dust environments not only induces profound bal fluid neutrophilia and airway obstruction in horses susceptible to severe asthma, but also significant neutrophilic airway inflammation in previously healthy horses ( , ) . outside of the experimental exposure setting, higher dust exposure has also been associated with increased risk of tracheal mucus accumulation in racing thoroughbreds ( ) . barn dust is a complex mixture, rich in potential sources of allergens as well as immunomodulators such as endotoxin and β-glucan ( , ) . in addition to individual horse factors such as age and susceptibility, this complexity may partially account for the heterogeneity of asthma phenotypes. respirable particulates, nominally < µm in diameter, have been linked to eosinophilic inflammation in young thoroughbreds entering race training ( ) and neutrophilic inflammation in actively racing thoroughbreds ( ) . increasing respirable endotoxin exposures have been shown to provide an apparent protective effect against neutrophilic inflammation at low doses ( ) , while high doses of endotoxin augment the inflammatory response to particulates ( ) , suggesting a non-linear response to inhaled endotoxin in the horse. mast cell inflammation has been found to be common in both young, untrained thoroughbreds ( ) and those that are actively racing ( ) , but unrelated to respirable dust or respirable endotoxin exposures. instead, bal mast cell proportions are related with respirable β-glucan exposures. conversely, inhalable dust exposures have not been found to affect bal inflammatory cell proportions. thus, inhalable particulates, those nominally < µm in diameter, appear to be less relevant than respirable particulates in equine respiratory health. setting exposure recommendations will require better understanding of the dose-response to inhaled non-infectious agents across wider ranges of age, breed, and discipline through study designs that include both exposure and respiratory health outcome measures and utilize appropriate statistical tools to relate them. advanced characterization of respiratory health, such as investigation of alveolar macrophage function and bal fluid cytokine profiles, coupled with extensive exposure assessment is likely to offer valuable insight into ea pathophysiology and identify new targets for intervention. miniaturization of optical particle counters has rendered realtime breathing zone exposure measurements on the horse both affordable and technically feasible. finally, the equine airway is arguably most susceptible to particle penetration during athletic exertion due to large tidal volumes and extension of the head and neck, yet the exposures that horses sustain during exercise are largely unexplored. such measures of exposure are complicated by the air speed and turbulence generated at the breathing zone during such activity and will require specialized sampling strategies. neutrophils are key actors in host defense, migrating toward sites of inflammation and infection, where they act as early responder cells toward external insults ( ). however, neutrophils can also mediate tissue damage in various noninfectious inflammatory processes. airway inflammation is one of the primary characteristics of an asthma-affected horse's response to aeroallergens with neutrophilic bronchiolitis being the main lesion ( ) . the mechanism by which airway inflammation develops in ea is a multifaceted and dynamic process. current knowledge suggests that the inflammatory component of this disease results from a combination of both the innate and adaptive immune responses ( ) . generally, airway inflammation involves activation of pathogen-specific inflammatory cells, modulation of gene transcription factors, and release of inflammatory mediators ( ) . within the airways, neutrophils likely contribute to bronchoconstriction, mucus hypersecretion, and pulmonary remodeling by release of proinflammatory mediators, including the cytokines interleukins and , neutrophil elastase, reactive oxygen species, and neutrophil extracellular traps (nets) ( ) ( ) ( ) ( ) . oxidative stress in horses with asthma is evidenced by the increase in elastase and decrease in ascorbic acid concentrations in balf associated with neutrophilia secondary to exposure to organic dust ( ) . the pathogenic role of nets has been described for many infectious and non-infectious human diseases, including respiratory cases with a massive influx of neutrophils into the airways ( ) . excessive net release is particularly deleterious in lung diseases because nets can expand easily in the pulmonary alveolar space and cause lung injury. furthermore, nets and their associated molecules can directly induce epithelial and endothelial cell death ( ) . the mechanisms that regulate neutrophil functions in tissues are complex and incompletely understood and must be regulated with exquisite precision and timing. timely apoptosis of neutrophils is central to the resolution of inflammation; dying neutrophils are known to stimulate their own efferocytosis, inducing macrophagic transition from a pro-inflammatory to an anti-inflammatory profile ( ) . thus, dysregulated apoptosis and mechanisms of inflammation may play an important role in the pathogenesis of ea. the persistence of apoptosis-resistant neutrophils in the airways of horses with asthma may also impede timely neutrophil clearance and delay the resolution of airway inflammation. the discovery and development of compounds that can help regulate ros, net formation, cytokine release and clearance of airway neutrophils would be highly beneficial in the design of therapies for ea ( ) . asthma is a highly heterogeneous condition of the lung. akin to the lining of the gastrointestinal tract, the lining of the airways is also in contact with external substances throughout life. ingested substances generally pass through the gastrointestinal tract unidirectionally, and a careful balance between processing of digested food materials, nutrient absorption and limiting immunoreactivity is maintained during homeostasis, with wellknown severe consequences of deviations in this balance. the airways function differently in that only gaseous substances normally pass into the distal alveoli and are exhaled in the reverse direction. inhaled particulates also have to be expelled in reverse direction toward the nasopharynx by largely mechanical means or taken up by alveolar macrophages for disposition with minimal inflammatory evocation ( ) . hence, a complex and selective epithelial barrier with differing functions characterizes both organs. the epithelium lining the airways has unique composition, morphology and function throughout the lung, and is intimately connected to subepithelial structures such as the basement membrane, mucous glands, smooth muscle, fibroblasts, endothelium and immune cells. the epithelium forms a barrier between inhaled components and the subepithelial constituents, and also has to balance efficient transfer of gases with controlled reactivity to non-gaseous components. while the lesions of severe ea manifest predominantly with inflammation, smooth muscle hyperplasia and fibrosis of the peripheral airways and surrounding tissues, the larger airways are exposed to the same inhaled substances and also have morphological, functional and molecular changes ( ) . research initially focused on the role of club cell secretory protein (ccsp), a member of the secretoglobin family produced by non-ciliated epithelial cells concentrated within the epithelium at the transition from bronchi to bronchioles. club cells are recognized as epithelial progenitor cells that can differentiate into ciliated and other specialized cells of the airway epithelium, participate in reduction of reactive oxygen toxicants through cytochrome enzymes, and their hydrophobic secreted protein inactivates a range of inflammatory mediators. horses with severe asthma have fewer club cells and lower concentration of ccsp in airway fluids, which may be a function of chronic inflammation resulting in reduced regenerative capacity of the airway epithelium ( ) . unique relative to other mammals, equids have two expressed ccsp genes that differ in of amino acids, and also in their interaction with hydrophobic molecules ( ) . recombinant eccsp increased neutrophil oxidative burst, phagocytosis and extracellular trap formation, lending support to the notion that loss of club cells has deleterious effects on lung health ( ) . whole transcriptomic changes in endobronchial epithelial biopsies from sites from th to th generation bronchi were investigated with next-generation sequencing. each horse served as its own control to identify changes in gene expression associated with an inhaled challenge since inter-individual variability exceeded changes attributable to the challenge. a bioinformatics pipeline including quality control measures to account for duplicates, variable sequencing depth and dispersion was implemented, results were mapped to the equine genome, and predicted proteins were procured with a combination of software and manual approaches to assign appropriate ensemble ids for analyzing interactions. an overall conservative analytic approach yielded genes differentially expressed in horses with severe asthma as a result of a challenge, with the majority up-regulated ( ) . not surprisingly, many up-regulated genes pertained to inflammatory mediators and effectors and were well-known members of protein interacting networks. however, somewhat more surprisingly, genes with altered expression also concerned more broadly epithelial cell formation and maintenance, and the circadian rhythm, suggesting that multiple cell properties are affected in exacerbated ea at the transcriptomic level. subsequent analysis of enriched gene sets in asthmatic horses further highlighted the importance of cell cycle regulation and repair pathways ( ) . transcriptomic studies of this nature yield a great deal of information, which requires subsequent confirmation regarding cell specificity, correlation with protein expression and function, and extension to a more robust number of affected and unaffected individuals. albeit, there is strong evidence to indicate that the bronchial epithelium is profoundly altered during exacerbation of severe ea, and this insight offers new venues for investigating the role of specific proteins and for potential therapeutic targets ( , ) . the entire spectrum of ea is influenced by interactions between the environment and genetics, but almost all research in this field has focused on the severe clinical phenotype. while no specific genetic risk factors have been reported for mild to moderate forms of ea, genetic susceptibility to certain bacterial lower airway infections could potentially be relevant ( ) . furthermore, mild but persistent respiratory signs such as occasional coughing and nasal discharge may represent early phenotypic indicators for an increased risk to later development of severe ea ( ) . this suggests that the genetics of milder forms of ea may be worth investigating in longitudinal studies. severe ea has been shown to be partly heritable in several breeds and has been the focus of genetic research involving family and epidemiological studies, whole-genome scans and investigation of candidate genes. reports of marked familial aggregation of severe ea date back years ( ) . parent, age, and stable environment have significant additive effects that increase the risk for developing severe ea as defined by a history of persistent frequent coughing and/or increased breathing effort ( , ) . offspring of affected sires have a more than -fold increased risk for developing severe ea ( ) . whole genome scans in high-prevalence families indicate two chromosome regions with a genome-wide significant association with severe ea ( ) . importantly, the associations differ between the families: region eca in one family and eca in another family. further association and gene expression studies indicate interleukin receptor as a candidate gene in a subset of ea-affected horses. molecular pathway analyses of genomic and proteomic data showed interactions between interleukin receptor and socs upstream of an important molecular cascade involving nuclear factor κb ( ) . so far, no causal genetic variant has been identified in interleukin . an allelic case-control genome-wide association study in the general warmblood population revealed another region on chromosome . the best-associated marker was located in the protein-coding gene txndc , which may be involved in regulating hydrogen peroxide production in the respiratory tract epithelium as well as in the expression of muc ac mucin ( ) . no genomic copy number variations were found to be associated with severe ea ( ) . integrative analyses combining gwas, differential expression (de), and expression quantitative trait loci (eqtls) were not able to uncover causative genetic variants that contribute to severe ea through gene expression regulation. however, results showed interesting similarities to human asthma with disease-associated genetic variants in clec a that also regulate gene expression of dexi ( ) . furthermore, global gene expression studies of mrna and mirna levels in these high-prevalence families have shown impaired cell cycle regulation and cd + t cell differentiation into th /th cells, respectively, in severe ea ( , ) . at present, none of these associations are useful genetic markers in the general population. most of the findings pertain to warmbloods only, or even only to certain lines and families. the fact that the chromosomal regions and the mode of inheritance do not agree between families indicates genetic heterogeneity for severe ea: depending on the genetic make-up of affected horses, different genes confer the susceptibility for the disease. it appears that the genetic basis of severe ea is robust, but remarkably complex. polygenic complexity, potentially with a larger number of genes that each may only contribute < % to the total genetic effects, may make it difficult to discover causative variants. nevertheless, the genetics of severe ea has revealed interesting links between severe ea, allergic skin diseases and susceptibility to intestinal parasites ( , ) . according to the national institutes of health, a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacologic responses to a therapeutic intervention ( ) . in practice, biomarkers include tools and technologies that can help in understanding the prediction, cause, diagnosis, progression, and outcome of treatment of a disease. although bal cytology has been recognized as the gold standard for diagnosing respiratory diseases such as ea, currently, sensitive and specific biomarker tests useful in routine laboratory diagnostics are being sought. a simple biomarker capable of distinguishing between animals with lower airway infections and those with non-infectious airway inflammation would be helpful. although the diagnosis of severe cases of ea is relatively easy, it is difficult to diagnose cases in remission or horses with a mild form of the disease. ideally, molecular biomarkers should reflect a feature of relevant pathological processes. in addition, biomarker assessment should be easy, low-cost, technically accurate, repeatable and have an acceptable risk. therefore, a measurement from easily obtainable body fluids or tissues is preferred, such as blood, urine, exhaled breath condensates, as opposed to bal, transbronchial biopsy or lung biopsy ( ) . several biomarkers are present or altered in the airways or circulation of horses with asthma. inflammatory markers such as acute phase proteins and cytokines have been studied as markers of systemic inflammation. however, the available literature on markers of systemic inflammation in horses with severe ea is not well-characterized and controversial ( , ( ) ( ) ( ) . apart from reports on differential expression of cytokines during the course of severe ea, only a few acute phase proteins have been investigated. haptoglobin is a suitable marker of both acute and chronic systemic inflammations, whereas high concentrations of serum amyloid a indicate acute inflammation. one study found no difference in the acute phase protein levels (serum amyloid a, c-reactive protein, haptoglobin) between horses with mild ea and those with other causes of exercise intolerance ( ) . another study found elevated haptoglobin concentration in horses with mild ea ( ) . surfactant protein d is a large multimeric collagenous glycoprotein produced mainly by type ii epithelial cells in the lungs and is also detectable in the serum. serum surfactant protein d has been identified as a potential systemic biomarker for some pulmonary diseases in humans, such as idiopathic interstitial fibrosis and acute respiratory distress syndrome. elevated serum levels of surfactant protein d have been detected in horses with mild ea ( , ) . circulating immune complexes are proteins that result from an immune response against an organism or antigens of various origin. in humans, circulating immune complexes are detectable in a variety of systemic disorders such as autoimmune diseases, allergies and infectious diseases ( ) . high levels of circulating immune complexes have been reported in horses with severe ea ( ) . another study found circulating immune complexes useful for differentiating healthy vs. severe ea, and monitoring corticosteroids therapy ( ) . the main group of enzymes responsible for collagen and other protein degradation in the extracellular matrix are matrix metalloproteinases (mmps), while tissue inhibitors of metalloproteinases (timps) lead to fibrosis formation. collagen is the main structural component of connective tissue and its degradation is a very important process in development, morphogenesis, tissue remodeling, and repair. in horses with severe ea, mmps, timps, and their ratios are useful in the evaluation of the severity of respiratory disease and in identifying subclinical cases ( ) . furthermore, mmp- , mmp- , timp- , and timp- are significantly decreased after therapy with inhaled glucocorticoid therapy ( ) . exhaled breath condensate is a promising source of biomarkers of lung disease in humans. exhaled breath condensate hydrogen peroxide concentration and ph were higher in horses with mild ea, vs. controls ( ) . additionally, both hydrogen peroxide and ph had a positive association with bal neutrophil percentage, while leukotriene b- demonstrated a positive association with bal eosinophil percentage. another study characterized the metabolomic profile of tracheal wash and exhaled breath condensate in healthy horses and those with severe ea ( ) . higher concentrations of histamine and oxidant agents, such as glutamate, valine, leucine, and isoleucine, as well as lower levels of ascorbate, methylamine, dimethylamine and o-phosphocholine, were found in the group of severe ea, compared to healthy controls. many biomarkers of ea have been studied-some are already being used in clinical settings, while others require further studies. however, history, clinical evaluation, and bal still constitute the basis for diagnosis of ea. immune response has mainly been investigated in the airways of horses with severe ea and more recently mild-moderate ea, while still representing one of the futures direction for research stated in the acvim consensus statement ( ). such characterization has mostly been performed through relative mrna expression of various cytokines in bal fluid, while several publications also reported protein concentration in bal fluid for few cytokines. various methodologies for cytokine mrna expressions have been published (e.g., sybr green or taqman technology, design of primers and probes, relative quantitation, etc.). variation in methodologies may ultimately prevent objective comparisons between reports, as well as the implementation of prospective, multicenter studies. such diversity should however not be considered as a scientific weakness, and methodological homogenization among the various research groups neither represents a prerequisite nor a final goal to be reached. however, evaluation of the methodological performances of different research laboratories might represent a relevant goal. in this manner, implementation of inter-laboratory comparisons based on international standards (e.g., iso/iec and iso ) warrants further consideration. let's consider for example mrna expression of two different cytokines by pcr in balf samples. as a first and informal procedure, a simple "blind test" could be performed among up to four different teams. in this procedure, the "reference lab" will provide the three other labs with aliquots of the same sample(s). each team will evaluate mrna expression for these two cytokines based on their own procedures, and comparisons of the results obtained and agreement among the teams can be evaluated. this "blind test" might then be repeated on a regular basis, systematically alternating the "reference lab" within the group. in the end, the procedure will provide an objective evaluation of the results diversity among the teams, but clearly will not determine whether several teams are more efficient than others for these specific analyses. a second and more structured procedure would require the specific synthesis of standards (mrna for two different cytokines in this case), and the development/validation of relevant conditioning and conservation procedures. a similar group of four different labs would first evaluate their ability to detect and quantify predetermined amounts of analytical standards (evaluation of the detection, not of the sample extraction, etc.). this step is a necessary preliminary, in the absence of reference methods. a panel of at least samples (previously calibrated with standards) would then be tested, including several identical ones (for repeatability) and submitted to the group (including a "self-shipment") for testing and further statistical analyses (agreement, etc.). once the methodological performance of the lab is considered acceptable for this panel, the procedure might then be repeated with another two cytokines and so on. in the end, the whole panel of standardized samples might allow the establishment of a labeling, accessible to any voluntary laboratory involved in equine asthma. mandatory considerations about such comparisons are that there is no trap, and this does not represent an overall examination of laboratories, but simple evaluations of procedures. all labs are expected to use their methodologies, whether or not the technologies are similar within the group. among others, samples conditioning, conservation, shipment and their associated costs will represent major issues to be considered, and this should be more broadly associated with virtuous initiatives such as the equine respiratory tissue biobank. several group discussions were conducted during the havemeyer equine asthma workshop to identify future research priorities. initial rotating small-group topic explorations (pathophysiology, risk-factors, diagnostic methods and phenotype definition) facilitated by members of the workshop organizing team, were followed by a final large group "roundtable" discussion of key directions for future ea research. the discussion was informed by data gathered directly from ∼ participants (i.e., all who attended the final roundtable), who were invited to propose up to three short-or long-term, focused or "big picture, " research topics or ideas that they considered to be key future research directions. these data were submitted anonymously, during the workshop, as free-text on paper and loosely arranged into broad categories for further open discussion. following the workshop, in order to present an accessible, systematic and non-selective summary of the ideas proposed by participants, the free-text data were collated in microsoft excel for content analysis using an approach based on recommended methods for quasi-qualitative data ( , ) . the text was transcribed verbatim and coded at two levels to categorize content into (i) broad topic areas (level ) and (ii) specific subsets of these topics (level ). all instances of each level topic code were then exported into online software (worditout) to create a word cloud (figure ) , in which the relative frequencies of occurrence of each topic are represented by font size. overall, responses were received, each proposing between and research ideas, resulting in a total of research ideas, which were organized into the broad topic codes presented in the word cloud. some research ideas encompassed more than one topic and were identified with multiple codes to reflect this. frequencies of occurrence of each code ranged from n= for "diagnostics" to n = for "genetics." specific proposed areas of interest in the dominant "diagnostics" category were the development of improved, non-invasive field diagnostics through the identification of suitable biomarkers, development of portable lung function tests, improved understanding of relative values of tracheal wash in comparison with bal cytology, or relationships between the two, and identification of gold standards for all of these diagnostic modalities. another key topic was phenotype distinction ( occurrences)-in particular to clarify any distinction between mild and moderate ea, and to determine whether or not such a distinction is valuable in terms of differing pathophysiology, diagnostic indicators, therapeutics or prognosis. as with many of these proposed topics, phenotype distinction rests on the back of the category "diagnostics"-pointing out a self-identified weakness on the part of ea researchers that the goal of identifying the horse with asthma so mild that it does not present as respiratory disease per se, continues in many cases to elude us and underscores a collective pragmatism that there is little benefit in understanding the fine points if we cannot definitively identify the case in the first place. ideas relating to therapeutics ( occurrences) included investigating the efficacy of different treatments including environmental management and any evidence for the value of antibiotics, as well as the development of optimal nebulized glucocorticoids, alternatives to corticosteroids, immunological treatments, respiratory probiotics, other novel therapeutics (e.g., marcks inhibitor peptide), and individualized treatments for different endotypes and phenotypes. suggestions relating to pathophysiology ( occurrences) included furthering our understanding of the role of environmental pollutants, of when a physiological response becomes a pathological response and of factors influencing progression from mild to severe equine asthma. standardization ( occurrences) referred in particular to the need to develop or agree on standardized diagnostic approaches, including in relation to bal collection techniques, laboratory processing and cytological methods and threshold values, context-specific reference ranges, development of a central repository of protocols and improved quality control protocols. a central repository of standard protocols was suggested. academic-clinical communication ( occurrences) was recognized as an area for general improvement. related research suggestions included improving our understanding of the views and practices of field clinicians, as well as their perceptions of disease progression and treatment efficacy, particularly in regions outside the uk (to build on the kinnison and cardwell uk study) ( ) . this would inform the enhancement of multidirectional communication between academia, referral and first opinion clinical practice, development of guidelines and apps for field practice and overall improved dialogue and engagement. better use of collaborative, epidemiological and longitudinal studies was suggested for many topics and included multicenter, cross-country collaborations, more use of the existing tissue bank and the initiation of a new equine asthma group. it is recognized that the ideas for research directions generated through this roundtable discussion at the end of a day workshop are subject to biases and influences relating to the interests, priorities and perceptions of workshop participants. however, by using and describing a systematic method of representing the ideas proposed, we have aimed at least to be transparent in our reporting of this. further, longer-term, international discussion and exchange of views will be facilitated by one of the key outcomes of this workshop, which was the development of the new equine asthma group. the aim of this group is to offer a platform of information for veterinary practitioners and horse owners as well as a resource for researchers to collaborate and exchange ideas on the understanding of ea. it was suggested that this group could lead some initiatives in line with the proposed areas of interest described above. there are plans for this group to develop some guidelines for the diagnosis and treatment of equine asthma, including for example the standardization of diagnostic methods, as mentioned above. development of an equine asthma group website and other communication tools are now underway as an internationally collaborative initiative. the havemeyer equine asthma workshop has paved the way for a better understanding of this many-faceted disease by bringing together researchers and clinicians to identify both the needs of the equine industry for effective treatments and at the same time focus researchers on the gaps in knowledge and understanding that will facilitate our ability to deliver on these needs. the participants made clear the requirement for more accessible, standardized diagnostics that will enable us to understand the underlying pathophysiology and identify specific phenotypes and endotypes and thus create more targeted treatments or management strategies. by creating an 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asthmalike disease of horses markers of systemic inflammation in horses with heaves circulating immune complexes and markers of systemic inflammation in rao-affected horses acute phase proteins in racehorses with inflammatory airway disease serum surfactant protein d and haptoglobin as potential biomarkers for inflammatory airway disease in horses tests for circulating immune complexes circulating immune complexes in horses with severe equine asthma metalloproteinases and their inhibitors are influenced by inhalative glucocorticoid therapy in combination with environmental dust reduction in equine recurrent airway obstruction metabolomics of tracheal wash samples and exhaled breath condensates in healthy horses and horses affected by equine asthma is there anything else you would like to tell us" -methodological issues in the use of free-text comments from postal surveys working with words: exploring textual analysis in medical education research the authors are grateful to gene pranzo, president of the havemeyer foundation, for his support of the workshop on equine asthma. we are also thankful to boehringer ingelheim animal health, haygain, nortev, trudell medical international, and zoetis for their support of travel grants for participants and workshop activities. the workshop was made possible thanks to the generous support of the dorothy havemeyer foundation, boehringer ingelheim, haygain, nortev, trudell medical and zoetis. key: cord- -ce ic g authors: sakata, kenneth k.; klassen, christine l.; bollin, kathryn b.; grys, thomas e.; slack, james l.; wesselius, lewis j.; vikram, holenarasipur r. title: microbiologic yield of bronchoalveolar lavage specimens from stem cell transplant recipients date: - - journal: transpl infect dis doi: . /tid. sha: doc_id: cord_uid: ce ic g purpose: stem cell transplant (sct) recipients commonly undergo bronchoalveolar lavage (bal) collection as an infectious pulmonary work‐up. previous studies report the utility and overall diagnostic yield of fiberoptic bronchoscopy with bal in this vulnerable population, though none focused purely on microbiologic yield or made comparisons with less invasive means of pathogen detection. we sought to determine and elaborate on the microbiologic yield of bal in sct recipients, assess a correlation between bal studies and less invasive means of pathogen detection, and assess the utility of repeating a bal within days. methods: between january , , and july , , we reviewed medical records of sct recipients who underwent bals. in addition to demographic information and details pertaining to their sct, a comprehensive review of their microbiologic data was performed and recorded. results: our study showed an overall bal microbiologic yield of %, despite % of patients receiving broad‐spectrum antimicrobial therapy at the time of the bal procedure. conclusions: although an initial bal sample in this population provides crucial microbiologic information, repeating the procedure within days may have minimal additional microbiologic yield. bal continues to be an essential diagnostic tool in sct recipients undergoing an infectious pulmonary work‐up. often requires both computed tomography (ct) and fiberoptic bronchoscopy (fob) with bronchoalveolar lavage (bal). fob with bal has generally been considered a safe and valuable procedure for evaluation of pulmonary complications in sct recipients undergoing an infectious pulmonary work-up. [ ] [ ] [ ] [ ] the bal specimen is evaluated with an array of diagnostic microbiologic studies targeting various pathogens, including bacteria, mycobacteria, fungi, and viruses. prior published studies have reported a diagnostic yield of bal ranging from % to %. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the data often combine the diagnostic yield of both infectious and noninfectious pulmonary complications in sct recipients. few reports focus exclusively on the microbiologic yield of bal in sct recipients. we therefore sought to determine and elaborate the overall microbiologic yield of bal in recipients of autologous (auto-sct) and allogeneic (allo-sct) scts, who underwent an infectious pulmonary work-up at our institution. the study cohort consisted of sct recipients who underwent bal procedures between january , and july , , at our institution as part of their infectious work-up for fever, respiratory symptoms, or new pulmonary radiographic abnormalities. for patients who had multiple bronchoscopy procedures, each encounter was considered as an independent procedure. patients who had an auto-sct before receiving an allo-sct were counted toward the allogeneic dataset. the ich bal order set at our institution during the study period con- the following data were collected for each bal ( %) identified a true pathogen respectively. the microorganisms isolated from bal samples from both auto-and allo-sct recipients are listed in table . of the true pathogens isolated, % were bacteria, % fungi, % viruses, and % were mixed organisms. of the bals in which a true pathogen was identified, % of the patients had received broad-spectrum antimicrobial therapy in the hours before bal collection. empirical antimicrobial therapy prior to bal was administered to % and % in the auto-sct and allo-sct subgroups respectively. the following pathogens were evaluated in more detail: pneumocystis jirovecii penicillium species saccharomyces species rhizopus species coccidioides species paecilomyces species trichoderma species bal, bronchoalveolar lavage; auto-sct, autologous stem cell transplant; allo-sct, allogeneic stem cell transplant; esbl, extended spectrum β-lactamase-producing. pneumocystis jirovecii, coccidioides species, and viruses. table summarizes the clinical information of patients with these pathogens. mrsa was isolated in bal cultures. fourteen bal cultures revealed pseudomonas- were collected from auto-and from allo-sct recipients. one of these had a concur- legionella was isolated in bal cultures- were retrieved from autoand from allo-sct. all had a concurrent positive bal legionella pcr. two cases were diagnosed within days of each other (one on the day of admission and requiring intensive care unit admission; the other, days later twenty bals were positive for aspergillus species- were from autoand from allo-sct patients. three different methods were used to assess for the presence of aspergillus: bal culture, bal aspag, and serum aspag. eight positive results were detected for bal p. jirovecii pcr- were detected from auto-and from allo-sct patients. all cases had a bal p. jirovecii fluorescent smear tested and tested positive. high-dose corticosteroid therapy was given in cases and cases received p. jirovecii prophylaxis ( pentamidine and trimethoprimsulfamethoxazole). the most common ct finding among the cases was diffuse ggo ( %). coccidioides species can be detected in the bal with fungal smear, culture, or pcr. there were positive cocci results on bal pcr, of which had a concurrent positive bal culture (and negative serum cocci serology). all positive bal cocci pcrs were from allo-sct patients. serum cocci serologies were tested in cases; had positive cocci serologic results, of which had a concordant bal pcr. the most common radiographic finding on chest ct was consolidations with ggo. the most common viruses detected were ebv (n= ), followed by parainfluenza virus (n= ; were isolated from auto-and from allo- fifteen bal specimens had or more concurrent pathogens, and the most common combination was bacterium and fungus (n= ), followed by fungus and virus (n= ) and by bacteria and virus (n= ). the other bal specimen isolated all of these pathogens. in this vulnerable population, fob with bal is considered to be a welltolerated, safe, and accurate procedure. complication rates of fob with bal in sct recipients can be as low as %. if transbronchoscopic lung biopsies are performed in addition to bal, complication rates can be as high as %- %. , the safety profile and low complication rate of bal make it the most commonly used diagnostic tool in sct recipients undergoing an infectious pulmonary work-up. administration of prophylactic or preemptive antimicrobial therapy is common in sct patients because empirical antimicrobial therapy is associated with improved clinical outcomes. however, early and aggressive use of such empirical regimens is conceptually thought to decrease the microbiologic yield of a bal sample. in our cohort of patients who had a true pathogen identified on bal, % had treatment with broad-spectrum antimicrobial therapy initiated at least hours before bal collection. in addition, % of our cohort with bals, who had detection of mrsa, pseudomonas, or aspergillus, or a combination of these, was taking appropriate therapy targeting these specific pathogens. therefore, receipt of concurrent antimicrobial therapy should not dissuade the operator from performing a bal in this patient population. mrsa pneumonia is associated with poor outcomes and frequently necessitates empirical antibiotic therapy. a recent study reported that a negative mrsa on pcr from a ns specimen had a % negative predictive value for mrsa pneumonia. we report similar findings wherein mrsa nss were performed and were negative. all bal and sputum cultures that were negative in these cases were negative for mrsa. in this series, of cases ( %) had a chest ct within hours of fob. chest ct scans may be more sensitive than chest radiographs, and ct is now the standard diagnostic tool in the initial assessment of invasive pulmonary aspergillosis and other opportunistic pulmonary infections in sct recipients. , this was a retrospective study and thus has its inherent limitations. we did not assess the impact of bal results on antimicrobial therapy (ie, alteration or initiation), morbidity, or death, because our study was designed only to analyze the microbiologic yield of bal. specific complication rates of fob were not collected. this also was a single-institution study and the results may not be generalizable to sct patients in other institutions or regions. to our knowledge, this is the largest retrospective study to date that looks at the microbiologic yield of fob with bal in sct recipients. we were able to demonstrate that sputum cultures are unreliable in detecting pulmonary pathogens in this population. in addition, obtaining a bal sample despite the patient receiving antimicrobial therapy for at least hours before sample collection continues to be a valuable diagnostic test to consider in this immunosuppressed cohort. bal is an essential diagnostic tool in sct recipients undergoing an infectious pulmonary work-up. our study showed an overall bal microbiologic yield of % despite % of this group having received empirical broad-spectrum antimicrobial therapy before bal collection. although an initial bal sample can provide critical microbiologic information, repeating a bal within days may not have additional diagnostic yield. k.k.s.: research concept/design, data collection, analysis, and interpretation, drafting article, critical revision of article, and approval of article; c.k.: data collection, analysis, critical revision of article, and data collection, critical revision of article, and approval of article data analysis and interpretation, critical revision of article, and approval of article research concept/design, data analysis and interpretation, critical revision of article, and approval of article pulmonary complications of bone marrow transplantation infectious pulmonary complications after stem cell transplantation or chemotherapy: diagnostic yield of bronchoalveolar lavage use of fiberoptic bronchoscopy in bone marrow transplant recipients retrospective utility of bronchoscopy after hematopoietic stem cell transplant the clinical importance of bronchoalveolar lavage in allogeneic sct patients with pneumonia utility of fiberoptic bronchoscopy in bone marrow transplant patients bronchoalveolar lavage in the diagnosis of pulmonary complications of bone marrow transplant patients role of bronchoalveolar lavage in the evaluation of interstitial pneumonitis in recipients of bone marrow transplants bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation utility of fiberoptic bronchoscopy in neutropenic patients admitted to the intensive care unit with pulmonary infiltrates investigation and management of pulmonary infiltrates following bone marrow transplantation: an eight year review pulmonary complications occurring after allogeneic bone marrow transplantation. a study of consecutive transplanted patients role of bronchoalveolar lavage in immunocompromised patients with pneumonia treated with broad spectrum antibiotic and antifungal regimen complications of fiberoptic bronchoscopy in thrombocytopenic patients infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults antimicrobial prophylaxis in bone marrow transplantation predictive value of methicillin-resistant staphylococcus aureus (mrsa) nasal swab pcr assay for mrsa pneumonia invasive aspergillosis treatment of aspergillosis: clinical practice guidelines of the infectious diseases society of america the use of respiratory-tract cultures in the diagnosis of invasive pulmonary aspergillosis film array respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples the role of chest ct in evaluation of the febrile bone marrow tranplant recipient high-resolution ultrafast chest ct in the clinical management of febrile bone marrow transplant patients with normal or nonspecific chest roentgenograms microbiologic yield of bronchoalveolar lavage specimens from stem cell transplant recipients the authors have no conflicts of interest to declare. key: cord- -vdm zvq authors: salton, francesco; geri, pietro; confalonieri, marco title: response to: factors limiting the utility of bronchoalveolar lavage in the diagnosis of covid- date: - - journal: eur respir j doi: . / . - sha: doc_id: cord_uid: vdm zvq we aimed at evaluating not the diagnostic yield of bal in covid- , but the agreement between negative upper respiratory tract swabs and bal to exclude covid- , stressing that bal is likely negative if swabs and chest ct are concordantly negative. francesco salton , pietro geri deepak and colleague misreported that bal was negative for sars-cov- rrt-pcr in majority of cases, including patients with "strong clinical and radiological suspicion for covid- ". indeed, we reported that, among those patients who underwent chest ct scan, revealed radiological signs compatible with an ongoing viral infection but not necessarily typical features of covid- . on the contrary, in the only cases of our series in which ct scan showed typical signs of covid- infection according to a recently published international consensus statement [ ] , bal was positive for sars-cov- despite previous negative upper respiratory tract swabs. moreover, we reported that, when chest ct scan was normal, then both upper respiratory tract swabs and bal were rrt-pcr-negative for sars-cov- . these findings support our main observation that bal is likely to be negative if one or more upper respiratory tract specimens and thoracic imaging are concordantly negative, therefore it should be only reserved for those cases in which a high clinical and radiological suspicion for covid- stands despite negative upper respiratory tract swabs. deepak and colleague also deem that our results might imply a high false negative rate of rrt-pcr for sars-cov- in bal samples. however, our study aimed at evaluating not the diagnostic yield of bal in covid- , but the agreement (test concordance) between negative upper respiratory tract swabs and bal to exclude covid- . this is a fundamental concept that we believe may be of valuable support to clinical practice in times of pandemics, when excessive demands for bal confirmation of repeatedly negative upper respiratory swabs expose operators to a high infectious risk while being clinically futile. in fact, bal has an unquestioned role in the diagnosis of pneumonia when non-invasive methods are not sufficient for an etiologic characterization; however, concerning sars-cov- , we showed that bal is most likely to be negative if upper respiratory swabs and chest ct are concordantly negative. we obviously agree with deepak and varinder that clinical performance of a diagnostic test varies not only in light of its sensitivity but also of pretest probability, that is affected by several factors among which disease prevalence. nevertheless, it is not possible to calculate a true pretest probability for covid- at the moment, as the actual prevalence of sars-cov- infection is still not known. [ ] finally, deepak and colleague claim insight details about our study population with regard to alternate diagnoses, clinical outcomes and their correlation with rrt-pcr test performance on upper and lower respiratory tract. unfortunately, this is out of the main focus of this study and it would need much more space than allowed by the journal for this manuscript format to be elucidated. the authors have no conflicts of interest to declare. we aimed at evaluating not the diagnostic yield of bal in covid- , but the agreement between negative upper respiratory tract swabs and bal to exclude covid- , stressing that bal is likely negative if swabs and chest ct are concordantly negative. limited role for bronchoalveolar lavage to exclude covid- after negative upper respiratory tract swabs: a multicenter study radiological society of north america expert consensus statement on reporting chest ct findings related to covid- . endorsed by the society of thoracic radiology, the american college of radiology, and rsna. radiol. cardiothorac. imaging false negative tests for sars-cov- infection -challenges and implications key: cord- -nt n p v authors: vissichelli, n. c.; miller, k.; mccarty, j. m.; roberts, c. h.; stevens, m. p.; de la cruz, o. title: bronchoalveolar lavage to evaluate new pulmonary infiltrates in allogeneic hematopoietic stem cell transplant recipients: impact on antimicrobial optimization date: - - journal: infection prevention in practice doi: . /j.infpip. . sha: doc_id: cord_uid: nt n p v summary background pulmonary complications cause significant morbidity and mortality after allogeneic hematopoietic stem cell transplant (ahsct). bronchoscopy with targeted bronchoalveolar lavage (bal) is often used in ahsct patients with suspected lower respiratory tract infection (lrti) to help guide management. aim to evaluate how positive bal results change antimicrobial management of ahsct recipients with suspected lrti. methods we performed a retrospective review of bal results from january to july for ahsct recipients. a positive bal was determined by culture, multiplex polymerase chain reaction (pcr), aspergillus galactomannan antigen (aga), and cytology. findings bal was positive for infectious etiologies in %, and antimicrobials were adjusted in / ( %) of patients. antibacterial escalation was predicted by a positive bal bacterial culture (or . , p= . ). antibiotic de-escalation was more likely with an elevated aga (or . , p= . ). antiviral initiation was more likely with positive bal multiplex pcr (or . , p= . ). antifungals were more likely to be escalated or changed with an elevated aga (or . , p= . ). the patients with a negative bal were more likely to be started on steroids (or . , p= . ). conclusions bal was helpful to determine the etiology of pulmonary complications and optimize antimicrobials. the addition of aga and multiplex pcr to standard bal significantly impacted de-escalating antibiotics and adjusting antifungals to provide adequate coverage. the association with an elevated aga with antibacterial de-escalation highlights a new role for bal in antimicrobial optimization. bronchoscopy with bronchoalveolar lavage (bal) is an important diagnostic tool to evaluate ahsct recipients with new pulmonary infiltrates or without clinical response to empiric therapy [ , ] . its utility is dependent on culture and molecular diagnostic results and the capability of changing medical management [ ] . in studies prior to the availability of polymerase chain reaction (pcr), antibiotics were withdrawn in up to % of ahsct recipients [ , ] . this study aims to determine how aspergillus galactomannan antigen (aga) and multiplex pcr added to traditional bal testing affects antimicrobial treatment in ahsct recipients with new pulmonary infiltrates. the first bal completed after ahsct in patients over age between january and july at the authors' institution were included. the decision to perform a bal was at the discretion of the attending physician caring for the patient. bal fluid was submitted for gram stain, bacterial, mycobacterial and fungal cultures, multiplex pcr, aga, and cytopathology. this study was approved by virginia commonwealth university's institutional review board. a positive bal was defined by a positive culture, multiplex pcr, elevated aga (optic density (od) > . ), and/or cytology (which includes detection of pneumocystis jirovecii). a positive bacterial culture required species isolation, excluding coagulase-negative staphylococcus and mixed respiratory flora. a positive fungal culture required the presence of fungal colonies, except non-cryptococcus yeast. the multiplex pcr used was the commercial kit biofire filmarrayÒ that can detect adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, influenzae a, influenzae b, parainfluenza (subtypes e ), respiratory syncytial virus (rsv), bordetella pertussis, chlamydophila pneumoniae, and mycoplasma pneumoniae. diffuse alveolar hemorrhage (dah) was diagnosed per guidelines [ ] . a change in management was defined by escalation or de-escalation of antibiotics, antifungals, or antivirals, adaptation of antifungals, or initiation of prednisone within days after bal since this is the time interval to obtain aga results at the authors' institution. deescalation was defined as cessation of one or multiple agents, conversion to oral therapy, or transition to a more narrow spectrum. escalation was defined as adding a new antimicrobial agent or transitioning to a broader spectrum. fungal adaptation was a transition to an alternative agent within the same class of antifungals. antimicrobial use within days of bal was considered prior therapy. ahsct was performed on the bone marrow transplant unit at the authors' institution. the conditioning regimen, immunosuppressive, and supportive therapies followed internationally accepted protocols. levofloxacin and fluconazole were used as anti-infective prophylaxis and prophylaxis for cytomegalovirus (cmv) and pneumocystis jirovecii pneumonia were used according to guidelines [ ] . a descriptive analysis performed using medians and interquartile ranges (iqr) for continuous variables due to nonnormal distributions and frequencies and percentages for categorical variables. we used a fisher's exact test to compare categorical variables and anova for continuous variables. a p-value of less than . was considered statistically significant. all analysis was performed using jmp pro [sas institute, cary, nc, usa]. bal was performed in ahsct recipients during the study period. procedures were excluded for patient age< (n¼ ), subsequent procedures on the same patient (n¼ ), and bal performed before transplant (n¼ ). patients who had a bal after ahsct were included. patient demographics, antimicrobials at bronchoscopy, and bal results are described in table i . most antimicrobial therapy was started within two days (n¼ ) or over days (n¼ ) before bal. eleven patients were not receiving antimicrobials. bal was positive for infectious etiologies in %, mostly with elevated aga ( / ), followed by multiplex pcr ( / ), positive bacterial ( / ), fungal ( / ) and afb culture ( / ). none of the patients with a positive bacterial culture were on levofloxacin prophylaxis. of the patients with a positive fungal result, / were on antifungal prophylaxis. only / patients with a positive bal aga also had a positive serum aga. all of the positive multiplex pcr were for viruses. nine patients had multiple infectious etiologies, all with a positive multiplex pcr. twentyeight patients had concomitant non-pulmonary infections. no patients were found to have pneumocystis jirovecicii, legionella, or cryptococcus by cytology or antigen testing. antimicrobials were adjusted in / ( %) of patients (table ii) . overall antibiotic escalation occurred in / , and was associated with a positive bal bacterial culture (or . , p¼ . ) (table ii) . antibiotic de-escalation was more likely with an elevated aga (or . , p¼ . ) (table ii) . antiviral initiation was more likely with positive bal multiplex pcr (or . , p¼ . ) (table ii) . antifungals were more likely to be escalated or changed with an elevated aga (or . , p¼ . ) ( table ii) . the patients with a negative bal were more likely to be started on steroids (or . , p¼ . ). bal was helpful to determine the etiology of pulmonary complications and optimize antimicrobials. this included escalating antibiotics to target organisms that were isolated. we believe it changes practice as bal is not only valuable when results are positive, but when an aggregate negative bal work up offers assurance for providers to address noninfectious etiologies. this is critical in intensive care settings when antiinflammatory therapy, such as high dose steroids, tumor necrosis factor inhibitors, or t-cell depleting agents are considered for pulmonary graft versus host disease, with suspected superimposed infection. only patients required initiation of targeted therapy for a microorganism isolated on bacterial culture that was not previously covered, including virulent organisms such as nocardia nova complex, pseudomonas aeruginosa, haemophilis influenzae, and stentrophomonas maltophilia. antibiotics were also escalated due to severity of illness despite negative culture data (n¼ ) and to cover an alternative infection (n¼ ), such as endocarditis. likely the use of antimicrobial prophylaxis effectively limited bacterial infections due to susceptible organisms, but may have added to the selection of more resistant bacteria such as stenotrophomonas and nocardia spp. bal was critical to recognize and treat invasive fungal infections, prompting antifungal escalation in % and initiation of targeted treatment for isolated rhizomucor spp. only two of the patients with an elevated bal aga also had an elevated serum aga. bal assisted in the diagnosis of these infections. this finding is important in that delays in treatment carry the potential for increased mortality [ ] . this is recognized in the american thoracic society guidelines, which suggest moving to bal aga and requesting bal aspergillus pcr when serum aga is negative. at a cutoff of . , the serum aga sensitivity is approximately e % and at . , it is e %. at a cutoff of . , sensitivity reduces to % and specificity increases to %. for bal aga with a cutoff of . , the sensitivity and specificity increases to % and % respectively [ ] . in addition, aspergillus spp. accounts for only % of invasive fungal infections in ahsct, and the remaining % includes deadly pathogens including zygomycetes ( e %), fusarium ( e % in usa), dematiaceous, and other rare molds that may require culture or tissue biopsy diagnosis. endemic mycoses only account for % of invasive fungal infections in ahsct and may be amenable to serologic or antigen testing without invasive procedures [ ] . a positive multiplex pcr was associated with antiviral initiation (ribavirin for parainfluenza or peramivir for influenzae), (table ii) . however, the results were similar on nasopharyngeal and bal multiplex pcr, as noted in prior studies [ ] . it is unclear if this multiplex pcr test is adequate for diagnosing lrti and more studies will need to be done to evaluate this. bal remained necessary to detect coinfections, as / patients with a positive multiplex pcr also had a positive bacterial culture (n¼ ) or elevated aga (n¼ ) ( table i) . reports of viral and bacterial coinfection, particularly with adenovirus or rhinovirus have been associated with higher morbidity/mortality but the understanding of this is limited [ , ] . it would be interesting to further investigate with larger studies the effect of viral illnesses on bacterial or fungal coinfections and how they affect treatment outcomes to determine if these more aggressive prophylaxis and diagnostic testing would benefit these patients. bal also had a major role in antimicrobial de-escalation. antibiotics were either stopped or converted to oral in patients with a negative bal bacterial culture. an elevated aga was predictive of antibiotic de-escalation (table ii) , which highlights a new role for bal in antibiotic optimization [ ] . a negative bal was associated with initiation of steroids for treatment of other conditions including graft versus host disease, pneumonitis, dah, and complications of engraftment (table ii) . even though the bal did not aid in these diagnosis (except dah), it mitigated the risk of starting steroids by lowering suspicion for pulmonary infections. study limitations include potential selection bias (only patients who had a bal were included) and the fact our results are from a single hospital. bal was performed at the discretion of the attending physician, and therefore the patient group only includes those deemed eligible for the procedure, which was likely urged by clinical status. a control group comparison (with or without available sputum cultures, nasal multiplex respiratory pathogen panel, serum aga and fungal urine antigen testing) would be likely biased to include those who were deemed less sick as well as those considered too high risk to be eligible for bronchoscopy, due to high oxygen requirement or bleeding risk. it would be unethical to randomly assign patients to bronchoalveolar lavage due to known risks and benefits. additional studies across multiple hospitals would be valuable to confirm our results. a multivariate regression analysis could not be performed due to low sample size. in summary, our study found that bal is a beneficial diagnostic tool to evaluate new pulmonary infiltrates in ahsct recipients. bal permitted targeted antimicrobial treatment and positively affected antimicrobial de-escalation and guided management of non-infectious diagnostic considerations. these data will inform local antimicrobial stewardship efforts. additional studies are needed to confirm our findings and to identify how bal can be used to improve antimicrobial prescribing. the authors declare that they have no conflict of interest or financial disclosures. diagnostic bronchoscopy in solid-organ and hematopoietic stem cell transplantation survey of academic pulmonologists, oncologists, and infectious disease physicians on the role of bronchoscopy in managing hematopoietic stem cell transplantation patients with pulmonary infiltrates role of bronchoalveolar lavage in the diagnosis of pulmonary infiltrates in immunocompromised patients bronchoscopic evaluation of pulmonary infiltrates following bone marrow transplantation the influence of diagnostic bronchoscopy on clinical outcomes comparing adult autologous and allogeneic bone marrow transplant patients diffuse alveolar hemorrhage guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective respiratory fungal infections in solid organ and hematopoietic stem cell transplantation microbiological laboratory testing in the diagnosis of fungal infections in pulmonary and critical care practice. an official american thoracic society clinical practice guideline filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples severe adenovirus pneumonia followed by bacterial septicaemia: relevance of co-infections in allogeneic hematopoietic stem cell transplantation human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients utility of early versus late fiberoptic bronchoscopy in the evaluation of new pulmonary infiltrates following hematopoietic stem cell transplantation the redcap software used in this effort is partially supported by ctsa award no. ul tr from the national center for advancing translational sciences. key: cord- -k kca zl authors: kamel, toufik; helms, julie; janssen-langenstein, ralf; kouatchet, achille; guillon, antoine; bourenne, jeremy; contou, damien; guervilly, christophe; coudroy, rémi; hoppe, marie anne; lascarrou, jean baptiste; quenot, jean pierre; colin, gwenhaël; meng, paris; roustan, jérôme; cracco, christophe; nay, mai-anh; boulain, thierry title: benefit-to-risk balance of bronchoalveolar lavage in the critically ill. a prospective, multicenter cohort study date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: k kca zl purpose: to assess the benefit-to-risk balance of bronchoalveolar lavage (bal) in intensive care unit (icu) patients. methods: in icus, we prospectively collected adverse events during or within h after bal and assessed the bal input for decision making in consecutive adult patients. the occurrence of a clinical adverse event at least of grade , i.e., sufficiently severe to need therapeutic action(s), including modification(s) in respiratory support, defined poor bal tolerance. the bal input for decision making was declared satisfactory if it allowed to interrupt or initiate one or several treatments. results: we included bal in patients [age years (interquartile range (iqr) – ); female gender: ( . %); simplified acute physiology score ii: (iqr - ); immunosuppression ( . %)]. bal was begun in non-intubated patients in ( . %) cases. sixty-seven ( . %) patients reached the grade of adverse event or higher. logistic regression showed that a bal performed by a non-experienced physician (non-pulmonologist, or intensivist with less than years in the specialty or less than bal performed) was the main predictor of poor bal tolerance in non-intubated patients [or: . ( % confidence interval . – . ); p = . ]. a satisfactory bal input for decision making was observed in ( . %) cases and was not predictable using logistic regression. conclusions: adverse events related to bal in icu patients are not infrequent nor necessarily benign. our findings call for an extreme caution, when envisaging a bal in icu patients and for a mandatory accompaniment of the less experienced physicians. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. bronchoalveolar lavage (bal) performed during fiberoptic bronchoscopy can help in diagnosing a vast array of lung diseases [ , ] . in the intensive care unit (icu), it is often performed in patients with acute respiratory failure. the main risk brought by fiberoptic bronchoscopy in the critically ill is the worsening of hypoxemia [ ] [ ] [ ] , but in the few studies focused on fiberoptic bronchoscopy tolerance and comprising a significant number of bal performed in icu patients, bal has been considered well tolerated in most of the cases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . meanwhile, as less invasive diagnostic methods exist or are emerging (high-resolution ct scan imaging, molecular microbiological diagnosis on nasopharyngeal swab, or on tracheal aspirates, etc.), the real utility of bal for the diagnosis of pulmonary diseases encountered in the icu may be questioned. in immunocompromised patients who represent a large proportion of patients undergoing bal in the icu, the diagnostic yield of bal was reported to be rather low compared to a less invasive approach [ ] . therefore, estimating the benefit-to-risk balance of bal in the critically ill would be an appreciable adjunct for decision making, when bal is envisaged. the objectives of this prospective, non-interventional, multicentre cohort study were to count and describe the adverse events observed during and after bal in the critically ill to estimate the proportion of patients for whom the bal fluid analysis allowed therapeutic decision(s) and to search for predisposing factors for either harm or benefit. the study took place in french medical-surgical icus (from public, university-affiliated [n = ] or non-university hospitals [n = ]) from april , to october , , complied with french law for observational studies, was approved by the comité de protection des personnes (approval number: . . ) and was registered with clinicaltrials.gov (nct ). patients or next-of-kin and physicians who performed the bal gave informed consent. patients were included if ( ) they had an indication to undergo a bal as decided by their attending intensivist, ( ) cellular analysis of bal fluid by a pathologist was planned, and ( ) consent had been obtained. pregnant women and patients under years of age were excluded. mini-bals, bals performed without bronchoscopy and bal without cellular analysis by a pathologist were not allowed. patients were included at time of their first bal during the icu stay. each center was asked to include at least patients and a maximum of patients. we planned to include patients and bals (see online resource for sample size considerations). data were recorded using paper case report forms filled in by local investigators and/or study nurses and then digitalized in the coordinating center (orléans). there was no on-site monitoring, but centers could be queried for clarification after centralized checking of data for completeness and consistency. the study was strictly non-interventional and physicians were asked not to modify their usual practice. we recorded the specialty (pulmonologist or intensivist) of the physician performing the bal. the physician's experience in terms of years in the specialty (< ; - ; > years) and of number of bal performed (< ; - ; > ) was recorded. we defined the physician performing the bal as an "experienced physician" when he/she was a pulmonologist or when he/ she was an intensivist with the greatest experience (i.e., > years in the specialty or > bal performed), considering that pulmonologists, by virtue of their specialty, are sufficiently trained in the practice of bal. we collected patients' characteristics at inclusion, including demographics, time spent in icu before bal, existence of acute respiratory failure before bal or not, according to the attending intensivist's judgment, simplified acute physiology score (sapsii) [ ] , tobacco use, underlying respiratory diseases, immunosuppression, and use of anticoagulant or antiplatelet therapy. we recorded the indication(s) of bal, vital signs before bal (respiratory rate [rr], heart rate [hr], blood pressure [bp]), and arterial blood gases, blood lactate, and pulse oximetry (spo ) within the past h. type of respiratory support used, body temperature, bp, hr, rr, and spo were collected at the beginning of bronchoscopy and at , , , and h, thereafter. the amounts of fluid instilled for bal and recovered were recorded. if sampled, arterial blood gases corresponding to the lowest pao /inspired fraction of oxygen (fio ) ratio within h after bal were also recorded. for patients under oxygen therapy other than high-flow nasal cannula oxygen therapy (hfnc), the fio value was derived from oxygen flow rate [ ] . in the critically ill, bronchoalveolar lavage (bal) is an aid for decision making in less than % of the cases and is associated with frequent, sometimes serious adverse events. adverse events and bronchoalveolar fluid of poor quality are observed more frequently, when bal is performed by the less experienced physicians. bal respiratory tolerance was first assessed by recording the need for modification(s) in respiratory support as previously described [ ] from the beginning of bronchoscopy to h after, including need of tracheal intubation, increase by more than % in oxygen flow rate, or use of hfnc in patients under standard oxygen therapy, increase by more than % in gas flow rate or fio in patients initially under hfnc, need of non-invasive ventilation (niv) in patients who initially had no mechanical respiratory support and increase by more than % in inspiratory pressure support or in positive end-expiratory pressure or in fio , in patients initially treated by niv. we added to this list the following events: increase by more than % in inspiratory pressure support or in positive end-expiratory pressure or in fio , or need of extracorporeal membrane oxygenation therapy in patients initially treated by invasive mechanical ventilation, and need for switching from pressure support mode to volume-controlled mode in patients with mechanical respiratory support. in addition, the investigators were asked to declare all clinically significant drops in spo and all other clinically significant events occurring during the h following the beginning of the bronchoscopy/bal procedure. all events were categorized in five grades of increasing severity (see table ). pathologists assessed the quality of the bal fluid. a bal fluid containing more than % of bronchial (squamous or ciliated epithelial) cells or less than , cells/ml or that was judged non interpretable for other reasons was said of "poor quality". otherwise, the bal fluid was considered of "good quality". the attending intensivists were asked, after having collected all analyses made on bal fluid, to categorize the bal according to the highest degree of usefulness it had reached: class , of no help; class , in line with (but not definitively confirming) a diagnosis already mentioned; class , suggesting a diagnosis not previously envisaged; class , allowing to interrupt one or several treatments; or class , bringing definitive diagnosis and/or allowing the initiation a new therapy. categorical variables are expressed as counts and percentages. continuous variables are expressed as median and interquartile range (iqr) or mean and sd. variables were compared between groups using χ test, fisher exact test, kruskal-wallis rank sum test, mann-whitney u test, one-way analysis of variance, or t test when appropriate. for multivariable analyses, missing values were replaced using multiple imputation by chained equations, and imputed datasets were pooled and analysed. multivariable logistic regressions with centers handled as random effect variable were used to identify predictors of "poor tolerance" (defined as the occurrence of at least one adverse event of grade or higher during the h-period of the study), and of "good usefulness" (degree of usefulness of class or ) (see online resource for detailed methods of variables/models selection). regarding bal tolerance, predictors were also searched in the framework of an ordinal regression model (proportional odds model) [ ] with mixed effects, using the highest grade of adverse event reached by each patient (as defined in table ) as an ordered categorical outcome. odds ratios (or) are given with their % the time courses of rr, hr, bp, and spo , from bal time (h ) to h after bal, were compared between types of respiratory support used at time of bal in the framework of distinct linear mixed models, adjusting for initial pao /fio ratio and sapsii, and patients handled as random effect. in these analyses, p values were adjusted for multiple comparisons using the tukey's test. all analyses were conducted using r software . . (http://www.r-proje ct.org). a two-side p value < . indicated statistical significance. among the bal performed during the study period, bal in patients were included (fig. other characteristics of patients and bal are exposed in table . a total of ( . %) patients needed modification of the respiratory support within the h after the beginning of bal, including eight ( . %) intubations in the hfnc/niv group and one ( . %) in the standard oxygen therapy group. percentages of patients needing change in the type of respiratory support are shown in table s online resource . a total of adverse events of any grade were observed (table ) . sixty-seven ( . %) patients reached the grade of adverse event or higher in the whole population. more patients in the hfnc/niv group ( (fig. ). in non-intubated patients, the percentage of grade adverse events was . % ( / ) when bal was performed by a non-experienced physician versus . % ( / ) otherwise, but the difference did not reach statistical significance due to small subsets sizes. logistic regression showed a strong interaction between the variables "invasive mechanical ventilation" and "experienced physician" (p < . ) (table s ). in the subset of non-intubated patients, a bal performed by a "non-experienced physician" was significantly associated with an increased risk of grade adverse events occurrence (or: . [ . - . ]; p = . ) (table s ) . logistic regression disclosed no significant predictor of grade adverse events in the invasive mechanical ventilation group (data not shown). proportional odds model analysis performed on the whole population also showed a strong interaction between the variables "invasive mechanical ventilation" and "experienced physician" (p < . ) (table s ). in the subset of non-intubated patients, a bal performed by a non-experienced physician was significantly associated with an increased risk of adverse events (or: . [ . - . ]; p = . ) and spo below % within h before bal, when entered as restricted cubic splines, placed the patients at risk of adverse events of grade or higher ( figure s ). no fig. study flow chart. a among the bronchoalveolar lavages (bal) performed during the study period, we did not record whether they comprised cellular analysis by a pathologist or if they were mini-bal or bal performed with or without bronchoscopy. b patient recruitment exceeded the expected, because we anticipated a number of non-workable case report forms h more than one indication could be present for each bal i significantly higher than in the nasal high-flow oxygen therapy or non-invasive ventilation group (p < . ), and then in the invasive mechanical ventilation group (p = . ) j h indicates the time at which bal has began k experience in years in the specialty and in terms of number of bal performed are detailed in table s of the online resource l we defined the physician performing the bal as an "experienced physician" when he/she was a pulmonologist or when he/she was an intensivist with the greatest experience (i.e., > years in the specialty or > bal performed) - . ]; p = . ) and the amount of bal fluid (in ml) recovered handled as a linear predictor (or . [ . - . ] per ml increase; p < . ), were statistically significant predictors of a bal fluid of good quality (table s ). transforming the amount of bal fluid in restricted cubic splines to take into account potential non-linearity gave a slightly better model fit and showed a biphasic relationship between the amount of bal fluid recovered and the probability of obtaining a bal of good quality (fig. ) . the same biphasic relationship was also founded in intubated patients ( figure s ), in non-intubated patients ( figure s ) , and in patients for whom the bal was not performed only for suspicion of hospital-acquired lung infection ( figure s ). no statistically significant predictor of a bal of good quality could be identified in patients for whom a suspicion of hospitalacquired lung infection was the sole indication of bal (data not shown). diagnoses retained for explaining the lung disease that justified the performance of bal are exposed in tables s , s , s , and s in the online resource . bal input was classified in class (not useful) in patients out of ( . %) ( counts and percentages of grade adverse event(s) during or after bal according to physician's experience and type of initial respiratory support. ns not significant. we defined the physician performing the bal as an "experienced physician" when he/she was a pulmonologist or when he/she was an intensivist with the greatest experience (i.e., > years in the specialty or > bal performed) there were / ( . %) bal classified as of good usefulness (i.e., class or ) in the whole population. this frequency was not statistically different between intubated and non-intubated patients at time of bal or between patients with bal performed only for suspicion of hospital-acquired lung infection or not (table s ). the quality of the bal fluid collected was not statistically associated with the bal usefulness: . % ( / ) of bal judged of good quality were classified in class or , versus . % ( / ) when bal was not judged of good quality by the pathologist (p = . ). multivariable logistic regression did not identify statistically significant predictors of a bal of class or (data not shown), either in the whole population or in pre-specified subsets. linear mixed model analysis showed that baseline and further spo values were lower in the standard oxygen group than in the two other types of respiratory support. other details of analyses are reported in figure s . in this multicenter cohort of critically ill patients, numerous adverse events were observed during or after bal and grade adverse events affected . % of the study population. the association of the noninvasiveness of respiratory support used with a bal performed by a nonexperienced physician was a strong predictor of adverse events occurrence. the experience of the physician performing the bal and the amount of bal fluid recovered were the main predictors of a bal fluid judged as of good quality by the pathologist. the bal input for decision making was satisfactory (i.e., allowed discontinuing a treatment and/or initiating a new one) in less than % of the cases. even when bal was indicated only for suspicion of hospital-acquired lung infection, a case where bal fluid quality might have less importance since most often mainly microbiological information is expected, the bal input was satisfactory in % ( / ) of the cases. interestingly, among the non-intubated patients with the most severe respiratory failure, for whom clinicians had judged standard oxygen therapy was not sufficient, only ( . %) had bal performed under niv, while the remaining patients had bal performed under hfnc therapy. in high-risk patients, niv for fiberoptic bronchoscopy with bal has been shown feasible [ , ] and recently, in one small-size randomized trial, safer than hfnc therapy [ ] . in counterpart, hfnc therapy has been shown safer than niv in patients with severe hypoxemic respiratory failure [ ] and has the advantage to be easy to use for care providers. this probably explains the predominant use of hfnc therapy in our study cohort. however, while some recent studies suggested that hfnc is safe for performing fiberoptic bronchoscopy and bal [ ] [ ] [ ] , large randomized trials are still needed. in the present study, adverse events collected were more frequent than in several previous studies [ ] [ ] [ ] [ ] ] . this discrepancy may be due to differences in methods used for collecting and defining adverse events. however, while the non-comparative design of this study does not allow firm conclusions regarding the cause-effect relationship between bal and the collected adverse events, the high frequency of adverse events should prompt caution when performing bal in icu patients, especially in non-intubated patients. the high frequency of adverse events might account for the increased hospital mortality recently observed in non-ventilated immunocompromised patients who underwent a bronchoscopy as compared to those who did not [ ] . given the high frequency of adverse events we observed in non-ventilated table s in online resource ) was used. the amount of bal fluid recovered was transformed in cubic splines to account for non-linearity. the biphasic shape of the figure shows that below ml of bal fluid recovered, the estimated probability declines in parallel with the amount of fluid recovered patients, one may wonder if systematically intubating the sickest patients (e.g., those with profound hypoxemia) could not be a safer option for performing bal. however, although this is a common concern in icus, to our knowledge, no comparative trial has yet been conducted to answer this question. it is worth noting that in the few available prospective studies focused on bal tolerance that showed rather low adverse event rate [ ] [ ] [ ] , bronchoscopies and bal were mostly performed by pulmonologists [ ] or by experienced icu physicians [ , ] . the present study highlights the importance of the physician's experience and training, which have often been emphasized in recommendations [ , , ] for bronchoscopy but have never been demonstrated for icu patients undergoing bal so far. although we could not show a direct link between the quality of the bal fluid recovered and the diagnostic yield of bal, the fact that the variable "experienced physician" was also a strong predictor of a bal of good quality suggests that the greater the experience of the physician performing the bal, the better the chance of performing a safe and useful bal. previous studies have reported that the diagnostic yield of bal was within the range of - % in icu patients [ , , ] . in line with these results, using a pragmatical classification in an unselected population, we found that the bal showed good usefulness for decision making in one half of the cases. additionally, we found that the bal input for decision making was not easily predictable. this uncertainty associated with the bal input for decision making again justifies the caution with which bal should be performed to ensure the best possible benefit-to-risk balance. although the bal usefulness of % observed in this study may appear rather low compared to the number of adverse events brought about, it should be placed in perspective with other diagnostic, invasive procedures such as open lung biopsy. in a recent meta-analysis in acute respiratory distress syndrome patients [ ] , open lung biopsy was reported to yield definitive diagnosis in nearly % of the cases, allowing to change therapy in %, while causing complications in %, resulting in a more favorable benefit-to-harm ratio than the one we observed for bal. however, great variability of diagnostic yield ( - %), impact on therapy ( - %) and complications ( - %) of lung biopsy existed between the published cohort studies [ ] , preventing any definitive conclusion. moreover, as those studies were mostly retrospective, it is highly probable that many adverse events were not collected. therefore, prospective studies that could help in deciding which procedure is riskier or more beneficial to patients are still lacking. in addition, although open lung biopsy may have proved beneficial to some immunosuppressed patients such as after bone marrow transplantation [ ] , it cannot reasonably be offered to all patients for whom bal currently appears to be the most appropriate diagnostic tool, either because it may be disproportionate for patients with low severity of disease or because surgical procedures are a real challenge for cancer patients with pancytopenia. undoubtedly, if there is one alternative to the bal that could be less risky for immunosuppressed patients, it is the combination of non-invasive tests on sputum, nasopharyngeal secretions, urine, and blood that may dramatically restrict the number of patients for whom bal is absolutely needed [ ] . this might become increasingly true in view of the current development of molecular diagnostic tools [ ] [ ] [ ] , the knowledge acquired in chest highresolution computed tomography imaging [ , ] , and perhaps future progress in the human volatilome analysis [ ] . this study has several limitations. first, because we did not study any control group in parallel, the adverse events collected cannot be attributed to bronchoscopy or to the bal with certainty. however, all the events collected are known side effects of bronchoscopy or bal and the close temporal relationship between bal and adverse events suggests that a non-negligible part of these events were related to the bal. second, we did not record the anesthesia regimen used during bronchoscopy and could not differentiate local from general anesthesia. some adverse events (e.g., hypotension) probably were side effects of intravenous anesthesia. anyway, our aim was to record, in real-life conditions, adverse events possibly related to the bal procedure, of which anesthesia is an integral part [ ] . conversely, the occurrence of adverse events such as hypertension, agitation, cough, or bronchospasm, at least in intubated patients, may reflect suboptimal anesthesia and leaves room for improvement. this suggests that in addition to an experienced physician performing the bal, the systematic presence of a second physician adjusting anesthesia, adapting ventilatory support and taking care of the hemodynamic status, might improve the patient safety. third, as there are no published specific classifications of adverse events and of diagnostic yield of bal, we used our own classifications. regarding the adverse events (table ) , it is noteworthy that the main predictor of adverse events identified (non-experienced physician performing a bal in a non-intubated patient), not only was strongly associated with grade adverse events, but also by proportional odds model analysis was associated with an increased probability of observing any grade of adverse event above a certain value versus observing any grade of adverse event below the same value. in our view, this would tend to validate the adverse events classification we proposed. fourth, we used a "home-made" classification of the bal input for decision making. this may have introduced bias in the estimation of the diagnostic yield of bal. in particular for the diagnosis of ventilator-associated pneumonia, it is possible that the culture of the bal fluid identified a microorganism already identified by other means (e.g., tracheal aspirate with semi-quantitative culture) and did not lead to modification of the antibiotic regimen already in place. although some may argue that in this case the diagnostic yield of bal might have been declared as very good, there is an ongoing debate about the bacteriological samples to be used for accurately diagnosing ventilatorassociated pneumonia [ , ] . also, in some instances despite no formal respiratory diagnosis was retained, the input of bal was classified as class (i.e., the best possible) by the attending intensivists, because bal not only has allowed ruling out some diagnoses, but also allowed trying another treatment such as diuretics or corticosteroids. this may have slightly distorted the classification of the bal input. fifth, in the french icus involved in this study, the bronchoscopy and bal could be performed either by pulmonologists or intensivists. therefore, our results may not be found in countries, where performing bronchoscopy is a prerogative exclusively reserved for pulmonologists. our findings suggest that in real-life conditions, adverse events during or following bal in icu patients are not infrequent nor necessarily benign. the lack of experience of the physician performing the bal was identified as the main predictor of clinically significant adverse events in non-intubated patients. on the other hand, the diagnostic yield of bal could be considered satisfactory in less than one half of the cases. altogether, these findings call for an extreme caution when considering the 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conflicts of interest in relation to this study. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.