key: cord-0893823-ysossker
authors: Scagnolari, Carolina; Midulla, Fabio; Selvaggi, Carla; Monteleone, Katia; Bonci, Enea; Papoff, Paola; Cangiano, Giulia; Marco, Paola Di; Moretti, Corrado; Pierangeli, Alessandra; Antonelli, Guido
title: Evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus
date: 2012-03-10
journal: Med Microbiol Immunol
DOI: 10.1007/s00430-012-0233-6
sha: df40b332d63d975f5c1fafc83809531da87294c7
doc_id: 893823
cord_uid: ysossker

The relationship between viral load, disease severity and antiviral immune activation in infants suffering from respiratory syncytial virus (RSV)-associated bronchiolitis has not been well identified. The main objective of this study was to determine the existence of a correlation between RSV load and disease severity and also between different clinical markers and mRNA levels of the interferon stimulated gene (ISG)56 in infants hospitalized for bronchiolitis. We also evaluated whether viral load tended to be persistent over the course of the RSV infection. The levels of RSV-RNA were quantified in nasopharyngeal washings, collected from 132 infants infected with RSV as a single (90.15%) or as a dual infection with other respiratory viruses (9.85%). Results indicated that viral load was positively related to the clinical severity of bronchiolitis, the length of hospital stay, the levels of glycemia and the relative gene expression of ISG56, whereas an inverse correlation was observed with the levels of hemoglobin. We also found that the RSV load significantly decreased between the first and second nasopharingeal washings sample in most subjects. These results suggest that infants with high RSV load on hospital admission are more likely to have both more severe bronchiolitis and a higher airway activation of antiviral immune response.

Bronchiolitis is an acute infection of the respiratory tract and is mainly associated with respiratory syncytial virus (RSV) [1] . The clinical spectrum of RSV bronchiolitis in previously healthy infants is extremely variable, ranging from mild upper respiratory symptoms to severe respiratory distress and, occasionally, death [2] . The factors determining severity of RSV-associated bronchiolitis have not been clearly established and are still unknown and are likely to be determined by a combination of host and viral factors. Host-derived risk factors associated with severe bronchiolitis include young age (<6 months), premature birth (<35 weeks of gestation), low birth weight, immunodeWciency or immunosuppression status, and congenital heart or chronic lung disease [2] [3] [4] . In addition single-nucleotide polymorphism in, and expression of, some genes codifying proteins involved especially in the control of innate immune response have been reported as being associated with the RSV disease severity [5] [6] [7] . On the other hand, the inXuence of viral factors such as the RSV-RNA levels on determining the bronchiolitis severity is not fully addressed. To this regard, some studies revealed signiWcant association between disease severity and RSV load in nasopharyngeal secretion of infants with primary respiratory tract infection [8] [9] [10] [11] . In contrast, other failed to found a correlation between bronchiolitis severity and viral load during primary RSV infection [12] [13] [14] .

Furthermore, a recent study found that the correlation between respiratory disease severity and viral load was the highest in case of single RSV infection but was absent in RSV coinfection, suggesting that infants with RSV as the primary pathogen infection were more severely ill [15] . Further, the clinical signiWcance of double infections of RSV and other respiratory viruses also appears to be unclear, and there are conXicting studies regarding the eVect of coinfection on disease severity [16] [17] [18] .

In the framework of a study aimed at understanding the pathogenesis of RSV infection and at further characterizing viral and host factors involved in determining the severity of bronchiolitis, we addressed whether any diVerences in RSV-RNA levels in the airway tracts of infants with bronchiolitis might explain the broad clinical spectrum of RSVassociated bronchiolitis. In addition, we evaluated the airway innate immune response by measuring the mRNA levels of gene coding for a cytoplasmatic antiviral protein, strongly induced in the lung after interferon (IFN) production or virus challenge [19] , namely the IFN stimulated gene 56 (ISG56). We, then, evaluated whether RSV load variations in bronchiolitis severity were correlated to level of ISG56-mRNA and whether viral load tended to be persistent over the course of the disease.

A total of 132 infants with a clinical diagnosis of RSVassociated bronchiolitis, admitted over four successive winter seasons (2006) (2007) (2008) (2009) (2010) to the Pediatric Department of Policlinico Umberto I Hospital, were included in this study. The institutional review board at Sapienza University of Rome approved the study. Written informed consent was obtained from the children's parents.

Bronchiolitis was diagnosed from the presence of a history of upper respiratory tract infection followed by acute onset of respiratory distress with cough, tachypnea, retraction and diVuse crackles on auscultation (wheezing alone was not considered suYcient cause for inclusion in the study). The exclusion criteria were underlying chronic condition (such as premature birth, cystic Wbrosis, chronic pulmonary disease, congenital heart disease, and immunodeWciency) and recurrent (more than one) wheezing episodes [17] . Disease severity and clinical evolution were evaluated using the clinical score index described by Midulla et al. [17] . In particular, on admission to hospital, a clinical severity score was assigned to each infant within the range 0-8, based on respiratory rate (<45/m = 0, 45-60/m = 1, >60/m = 2) arterial oxygen saturation in room air (>95% = 0, 95-90% = 1, <90% = 2), presence of retractions (none = 0, present = 1, present + nasal Xare = 2), and ability to feed (normal = 0, reduced = 1, endovenous = 2).

Nasopharingeal washings (NPW) were collected from 132 infants suVering from bronchiolitis in the Wrst 24 h after admission to hospital and an aliquot was tested for viruses as previously described [20] . A subset of 56 NPW samples was centrifuged at 2,000 rpm for 10 min and each cell pellet was resuspended in 1 ml of phenol and guanidine isothiocyanate reagent (Trizol, Gibco BRL, NY) and frozen at ¡80°C for subsequent ISG56 gene expression analysis.

PCR assays for respiratory viruses A panel of reverse transcription (RT) PCR or nested PCR assays, some in a multiplex format, was used for detection of fourteen respiratory viruses including: RSV, inXuenza A and B, coronaviruses, OC43, 229 E, NL63, HKU1, metapneumovirus, adenovirus, rhinovirus, parainXuenza 1-3, and bocavirus as previously reported [20, 21] .

TaqMan-based real-time RT-PCR technique for RSV detection A TaqMan-based real-time PCR technique for RSV-RNA quantiWcation was performed on all NPW specimens with positive RT-PCR results for RSV. BrieXy, viral RNA was extracted from NPW specimens that were positive for RSV using a QIAamp Viral RNA Mini Kit (Qiagen Spa, Milan, Italy). The RNA was dissolved in RNase-free water and the RSV quantiWcation was performed by Taqman assay after generation of cDNA using a High Capacity cDNA Archive Kit (applied biosystems, Monza, Italy). Type-speciWc primers and probes for N gene of both RSV A and B [22] were added to the universal PCR master mix (applied biosystems) at 300 and 100 nM, respectively, in a Wnal volume of 50 l. The standards were obtained by cloning the 82 bp of viral N gene into the pCR2.1 plasmid using a TOPO TA cloning kit (In Vitrogen Corporation, San Diego, CA, USA). A linear distribution (r = 0.99) was obtained between 10 1 and 10 8 copies of RSV-DNA. Viral load values were Log transformed for analysis and data was expressed as the Log number of RSV copies per ml of NPW.

The mRNA copy content of ISG56 was measured by a realtime 5Ј exonuclease RT-PCR assay using the ABI 7000 sequence detector (applied biosystems). BrieXy, the total cellular RNA was extracted from the cells using the Trizol reagent, following the manufacturer's instructions, and was retro transcribed as previously described [23] . Primers and probes for ISG56 gene were added to the universal PCR master mix (applied biosystems) at 300 and 200 nM, respectively, in a Wnal volume of 50 l [23] . Co-ampliWcation of the beta-glucuronidase gene (assay-on-demand, Hs99999908_m1, applied biosystems) was used to normalize the amount of total RNA present using the threshold cycle relative quantiWcation according to the supplier's guidelines.

Descriptive analysis was made using median (range) or frequency (percentage). RSV load values were expressed as Log copy number of RSV-RNA/ml and related geometric mean. DiVerences between infants with RSV as single or dual infection, in terms of the level of viral load, were compared using the Mann-Whitney test. Spearman's rho coeYcient was calculated in order to assess the correlation between the level of RSV load and the demographic and clinical parameters, and ISG56-mRNA levels measured in cells collected from NPW. The diVerences in the number of RSV-positive infants characterized by the presence or absence of retractions and/or nasal Xare according to the RSV load levels were evaluated using the chi squared test for trend. DiVerences in the RSV load between the Wrst and second NPW sample were compared using the Wilcoxon Test. SigniWcance was Wxed at the 5% level. The analysis was performed using SPSS v.13.0 for windows.

One hundred thirty-two infants diagnosed with bronchiolitis over a period of 4 years were included ( Table 1 ). The median age of the infants was 2.20 months, of which 78.69% had <6 months, and the sex ratio was 1. A total of 119 (90.15%) infants carried a single RSV infection whereas 13 (9.85%) had a coinfection with other respiratory viruses. The median clinical score level was 4; 22 (16.7%) infants had a severe bronchiolitis with a clinical score ¸7; 48 infants (36.4%) required oxygen supplementation and 37 infants (28.0%) had retraction with nasal Xaring.

An RT-Taqman assay was used to determine RSV load (Log copy number of RSV-RNA/ml) in NPW collected from all 132 infants with bronchiolitis. There was a distribution of viral load values of several orders of magnitude in infants with bronchiolitis (Fig. 1) . The Log copies of RSV-RNA per ml ranged from 1.68 to 8.93 and the median viral load values was 5.47. The distribution of RSV load showed that half of infants have a copy number of RSV-RNA per ml within the range of 5.47-8.93 Log. When the RSV-positive infants were divided on the basis of RSV detection as a single infection or as a coinfection, the viral load was not signiWcantly diVerent between groups (p = 0.49).

The relationship between patient data as independent variables and RSV-RNA levels measured in NPW was analyzed (Table 2) . A positive correlation between Log number of RSV-RNA copies/ml and clinical score index was found in infants with RSV infection (r = 0.17, p = 0.024). In particular, RSV load seems to inXuence the severity of respiratory disease. Indeed, when RSV loads were categorized into three groups ranked from lowest to highest values and analyzed in function of the presence or absence of retractions and/or nasal Xaring, we observed that the number of infants who have both the highest RSV load values (>6.84 Log) and the presence of intercostal retractions and nasal Xaring was higher with respect to the other groups (p = 0.020; Fig. 2 ). Furthermore, we found a signiWcant correlation between RSV viral load and the length of hospital stay (r = 0.16, p = 0.038), and levels of hemoglobin (r = ¡0.18, p = 0.024) or glycemia (r = 0.24, p = 0.028). In contrast, we failed to detect any correlation between the RSV-RNA levels and age or weight of tested infants, numbers of neutrophils, lymphocytes, eosinophils or platelets, and levels of sodium, or C-reactive protein ( Table 2) . Also no diVerence was detected in viral load for male and female and between infants with fever or without fever (data not shown).

In an attempt to determine whether RSV load inXuences gene expression of a well-known antiviral protein such as ISG56, levels of RSV-RNA were examined for any signiWcant correlation with expression of ISG56-mRNA. An RT-Taqman assay speciWc for ISG56 transcript was performed on the NPW samples from a subset of infants with single RSV infection (n = 56), for which we collected cells. A sig-niWcant positive correlation (r = 0.35, p = 0.008) was observed between viral load and levels of ISG56-mRNA (Fig. 3) .

We also evaluated changes in RSV load in a subset of NPW samples collected from 17 infants at the time of admission and at hospital discharge, after a median of 2 days (range 1-15 days). Fourteen had single RSV infection and 3 had double infection (2 metapneumovirus; 1 rhinovirus). Results indicated that RSV load decreased signiWcantly (p = 0.012) in most infants (71%) between the Wrst (median Log copies of RSV-RNA/ml: 5.95) and second NPW samples (median Log copies of RSV-RNA/ml: 3.22) independently of the presence of RSV as single or dual infection. In particular, a decline of RSV-RNA levels of at least 2.7 Log was observed in half of infants who have a viral load decrease, whereas in the remaining infants the Log copy number of RSV/RNA per ml decreased within the range of 1.76-0.40. In contrast no change in viral load, and an increase in copy number of RVS-RNA copies/ml within the range of 0.18-4.65 Log, were observed respectively in one and four infants.

This study showed that level of RSV load, measured shortly after hospital admission, was highly variable among infants suVering from bronchiolitis in agreement to several other studies [9, 10, 12] . In addition we found that coinfection of RSV and other respiratory viruses did not change signiWcantly RSV load although it has been reported that RSV levels were higher in infants with RSV as the primary pathogen that in infants with RSV coinfection [15, 18] . Interestingly, our results demonstrated a positive correlation between RSV load and the clinical score index used for determining the severity of bronchiolitis. In particular, we found that in infants with high levels of RSV-RNA in their NPW, there is a tendency to have more signs of severe respiratory distress. An association between high RSV load and the respiratory disease severity has been reported in previous papers [8] [9] [10] [11] , whereas other studies have failed to detect such an association [12] [13] [14] . Studies that did not Wnd this correlation may have been confounded by the presence in children of risk factors such as cardiopulmonary disease or of previous RSV infections, or by the use of diVerent molecular or virological techniques for RSV level evaluation [12] [13] [14] . The conXicting results could be also explained considering the diVerent bronchiolitis diagnostic criteria and/or clinical scores used in the various studies [8] [9] [10] 12] . Furthermore, we demonstrated that RSV load was positively correlated with the duration of hospital stay, an indirect indicator of the severity of RSV-associated illness. In agreement, other studies have reported higher nasal RSV load as a signiWcant independent predictor of longer hospitalization [9, 24] . However, in 23% of the infants in which we could measure RSV-RNA level at hospital discharge, an increase of viral load has been recorded.

In addition, our results indicate that viral load correlates with glycemic levels and hemoglobin values but not with other clinical characteristics such as C-reactive protein levels or leukocytes count as reported recently by Franz A and coauthors [25] . The mechanism by which RSV load aVects levels of glycemia and hemoglobin is currently unknown. However it has been reported that hyperglycemia is frequent in children with RSV infection [26] . Furthermore, recently anemia has been found to aVect the higher rate of thrombocytosis observed in infants with RSV-associated bronchiolitis [27] .

The apparent discrepancies along our data on the dynamic of RSV load in bronchiolitis, make more evident the complexity of the RSV-host interactions and suggest the need of evaluating also host factors for fully understanding the pathogenesis of RSV infection. Directly related to this, it is well established that the clinical spectrum of bronchiolitis might reXect diVerences in the proWle and extent of immunological and/or inXammatory mediators produced by RSV infected respiratory epithelial cells [6, 28, 29] .

Here, we demonstrated for the Wrst time to our knowledge that a high RSV-RNA level correlates with high levels of ISG56-mRNA in the airways of infants suVering from bronchiolitis. This protein has been implicated in the suppression of viral replication and protein translation and it seems also to be a mediator of negative-feedback regulation of virus-triggered induction of type I IFN and cellular antiviral response [30] . Interestingly it has been demonstrated that the nonstructural NS1 and NS2 proteins of RSV appear to antagonize both the cellular antiviral response as well as the induction of IFN transcription [31] [32] [33] . In addition Ling and coauthors have showed that NS2 inhibited ISG56 promoter activity induced by RNA-activated RIG-I in a dosedependent manner [34] . However, although RSV has evolved accessory gene products that neutralize or inhibit various steps of the IFN pathway, in our previous study we found a strong and a coordinated induction of IFN stimulated genes in the airway tract of infants with RSV infection [28] . Furthermore recently Pichlmair and coauthors have proposed that ISG56 protein antagonizes viruses by sequestering speciWc viral nucleic acids [35] .

Therefore our results suggest that quantitative diVerences in the replication of RSV may inXuence the magnitude of innate immune activation which in turn can determine the clinical course of bronchiolitis in either beneWcial or detrimental direction as previously suggested [5, 28] .

A potential limitation of this study was that we did not perform the evaluation of known pathogenic bacteria to explore whether the presence of bacterial superinfection aVects levels of RSV-RNA. Indeed, considering that infants hospitalized for bronchiolitis have low reported rates of serious bacterial illness [36] , we have collected only NPW and not well established respiratory samples such as those from lower respiratory tract, which are likely most suitable for performing isolation and identiWcation of bacteria. In addition, although the marked elevation of C-reactive protein observed in some of the RSV-positive infants could hints to bacterial superinfection, no signiWcance diVerence in leukocyte count as well as in chest X rays signs was found among infants who have elevated C-reactive protein levels and those with normal C-reactive protein values suggesting that globally there is no concomitant bacterial superinfection (data not shown). Furthermore, recently it has been demonstrated that C-reactive protein levels could be aVected by the presence of respiratory viral infection [25] .

In conclusion, our results showed that RSV load has a signiWcant impact on the clinical and immunological parameters of hospitalized infants suVering from bronchiolitis and underline the importance of implementing diagnostic tools for a rapid, sensitive and quantitative RSV detection in order to improve the clinical management of bronchiolitis. Further larger scale studies are needed to support the predictive value of measuring RSV load on the clinical course of bronchiolitis and to establish the actual "cutoV value" of RSV load associated to disease severity.

Respiratory syncytial virus and parainXuenza virus

Acute viral bronchiolitis in children-a very common condition with few therapeutic options

Susceptibility to bronchiolitis in infants

Incidence and predisposing factors for severe disease in previously healthy term infants experiencing their Wrst episode of bronchiolitis

Human genetic factors and respiratory syncytial virus disease severity

Viral and host factors in human respiratory syncytial virus pathogenesis

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Nasal quantity of respiratory syncytical virus correlates with disease severity in hospitalized infants

Respiratory syncytial virus load predicts disease severity in previously healthy infants

Respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity

Clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus

Illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus

Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis

Quantitation of respiratory viruses in relation to clinical course in children with acute respiratory tract infections

Disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community

Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis

Respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants

Multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children

Tissue-speciWc and inducer-speciWc diVerential induction of ISG56 and ISG54 in mice

Detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in

Human bocavirus infection in hospitalized children in Italy

Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR

The synergistic interaction of interferon types I and II leads to marked reduction in severe acute respiratory syndrome-associated coronavirus replication and increase in the expression of mRNAs for interferon-induced proteins

Correlation of viral load as determined by real-time RT-PCR and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy

Correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection

Glycemic level in mechanically ventilated children with bronchiolitis

Respiratory syncytial virus-positive bronchiolitis in hospitalized infants is associated with thrombocytosis

Upregulation of interferon-induced genes in infants with virusassociated acute bronchiolitis

Immunopathogenesis of respiratory syncytial virus bronchiolitis

ISG56 is a negative-feedback regulator of virus-triggered signalling and cellular antiviral response

Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages

Respiratory syncytial virus nonstructural proteins NS1 and NS2 mediate inhibition of Stat2 expression and alpha/beta interferon responsiveness

Respiratory syncytial virus nonstructural protein 2 speciWcally inhibits type I interferon signal transduction

Human respiratory syncytial virus nonstructural protein NS2 antagonizes the activation of beta interferon transcription by interacting with RIG-I

IFIT1 is an antiviral protein that recognizes 5Ј-triphosphate RNA

Bronchiolitis: in-patient focus

Acknowledgments This work was supported by grants to G.A from Pasteur Institute (Cenci Bolognetti Foundation; title of the project: "Molecular characterization of viruses causing bronchiolitis and study of viral and host factors aVecting Type I IFN antiviral response induced by respiratory viruses"), and to A.P. from "Sapienza" Università di Roma (Fondi ricerche Universitarie) and Italian Ministry of Health (Ricerca Finalizzata Conv. No. 88).