Acta Pædiatrica ISSN 0803–5253

REGULAR ARTICLE

Multiple viral respiratory pathogens in children with bronchiolitis

Hilary E Stempel1 , Emily T Martin1 , Jane Kuypers2 , Janet A Englund1,3 , Danielle M Zerr (zerr@u.washington.edu)1,3
1.Seattle Children’s Hospital Research Institute, University of Washington, Seattle, WA, USA
2.Laboratory Medicine, University of Washington, Seattle, WA, USA
3.Department of Pediatrics, University of Washington, Seattle, WA, USA

Keywords Abstract
Bronchiolitis, Respiratory syncytial virus,
Aim: The aim of the study was to describe the frequency of viral pathogens and relative frequency of
Viral infections
Correspondence
co-infections in nasal specimens obtained from young children with bronchiolitis receiving care at a
Danielle M. Zerr, 4800 Sand Point Way NE, R5441, children’s hospital.
Seattle, WA 98105, USA. Methods: We conducted a study of nasal wash specimens using real-time PCR and
Tel: +1-12069872653 |
Fax: +1-12069873890 |
fluorescent-antibody assay results from children less than two with an ICD-9-CM code for
Email: zerr@u.washington.edu bronchiolitis. All specimens were collected for clinical care at Children’s Hospital in Seattle, WA, USA,
Received during the respiratory season from October 2003 to April 2004.
10 April 2008; accepted 13 August 2008. Results: Viruses were detected in 168 (93%) of the 180 children with bronchiolitis. A single virus
DOI:10.1111/j.1651-2227.2008.01023.x was identified in 127 (71%) children and multiple viruses in 41 (23%). Respiratory syncytial virus
(RSV) was the most common virus detected (77%), followed by adenovirus (15%), human
metapneumovirus (11%), coronavirus (8%), parainfluenza (6%) and influenza (1%). Of the 139
samples with RSV detected, 34 (24%) were co-infected with another viral pathogen.

Conclusion: Molecular diagnostic techniques identified a high frequency of viruses and viral co-infections among
children evaluated for bronchiolitis. Further study of the role of viral pathogens other than RSV and co-infections
with RSV in children with bronchiolitis appears warranted.

sensitive diagnostic methods, such as polymerase chain reac-

INTRODUCTION
tion assays (PCR), has increased the number of viruses (e.g.,
Bronchiolitis is the most common cause of infectious dis-
human metapnuemovirus) detected in children with acute
ease hospitalization among infants in the United States (1).
respiratory tract infections (6–12). Most previous studies of
Approximately 3% of children under the age of 1 year
respiratory viruses in children using molecular diagnostics
are hospitalized annually for bronchiolitis, and the cost of
have focused on children with a spectrum of respiratory
hospitalization is estimated to exceed $700 million (2,3).
tract infections some of which have been associated with
Although bronchiolitis is a common disease that is readily
acute wheezing (7,8,12). There have been few population-
diagnosed clinically, the standard of practice for diagnosis
based studies of children with bronchiolitis using molecular
and management of bronchiolitis is still a source of debate.
diagnostics (10,11).
To address these concerns, the American Academy of
The objective of our study was to determine the relative
Pediatrics (AAP) developed and published a clinical prac-
frequency of various viral pathogens and the frequency of
tice guideline in 2006. This Guideline recommends that di-
viral co-infections in children less than 2 years with bron-
agnosis be based on history and physical examination, and
chiolitis presenting for acute care in a hospital setting.
discourages virologic testing on the basis that results rarely
impact management decisions. The Guideline states, how-
ever, that virologic testing may be useful when patient PATIENTS AND METHODS
cohorting is possible (3). This study was approved by the Institutional Review Board
Respiratory syncytial virus (RSV) is the predominate virus of Children’s Hospital and Regional Medical Center in Seat-
classically associated with bronchiolitis as reported in stud- tle, WA, USA. Residual clinical nasal wash samples were
ies that relied on culture and serologic detection methods collected from children of all ages presenting with respira-
(4). RSV is detected in 43-74% of cases of bronchiolitis, tory symptoms to the emergency department or inpatient
and parainfluenza (PIV), adenovirus and influenza are other at Children’s Hospital Regional Medical Center in Seattle,
commonly detected viruses (3,5). The development of more Washington between October 2003 and April 2004.
October through April encompasses the period of greatest
Abbreviations respiratory virus activity in our region. In total, 831 nasal
AAP, American Association of Pediatrics; PCR, polymerase chain wash samples with sufficient residual material were avail-
reaction; FA, fluorescent antibody; RSV, respiratory syncytial able for testing by real-time PCR as previously published
virus; PIV, parainfluenza; hMPV, human metapneumovirus; RV, (13). The current study includes a subset of these samples,
rhinovirus. specifically those obtained from patients less than 24 months


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Viral pathogens in bronchiolitis Stempel et al.

Table 1 Frequency of 210 individual respiratory pathogens detected by FA and/or real-time PCR in 180 children with bronchiolitis

Age Specific viral pathogens, n (%)1

RSV Adenovirus hMPV Coronavirus Parainfluenza Influenza No virus detected

<6 months (n = 83) 70 (84) 4 (5) 8 (10) 5 (6) 2 (2) 0 (0) 3 (4)
≥6 to <12 months (n = 51) 37 (73) 11 (22) 7 (14) 3 (6) 6 (12) 0 (0) 3 (6)
≥12 to <24 months (n = 46) 32 (70) 12 (26) 4 (9) 6 (13) 2 (4) 1 (2) 6 (13)
All ages n = 180 139 (77) 27 (15) 19 (11) 14 (8) 10 (6) 1 (1) 12 (7)
a
Percentages are reflective of pathogens detected within a specific age group; totals will exceed 100% because of co-infections.

of age with bronchiolitis. Billing records were obtained from Statistical analysis
patients with residual nasal wash samples. Bronchiolitis Statistical analyses were performed using Stata 9 (College
was defined by the International Classification of Diseases, Station, TX, USA). Percents, averages, medians and ranges
Ninth Revision, Clinical Modification (ICD-9-CM) codes were calculated for demographic variables and virologic test
for bronchiolitis (#466.11, #466.19). Based on this defini- results.
tion, 180 infants less than 24 months of age were included in
our study population. Each eligible patient contributed only RESULTS
one sample to the analysis. When a single child had multi- Single nasal wash samples from 180 children up to
ple hospital visits and/or samples, the earliest sample was 24 months of age with a diagnosis of bronchiolitis were
selected for this analysis. Clinical data were abstracted from collected between October 2003 and April 2004. Fifty-five
medical records using a standardized form. Demographic percent of the study subjects were male and the median
information including age, gender, and underlying diseases age was 6.5 months (range 0.4-23.5 months). The majority
or conditions were recorded. of children were admitted to a general pediatric ward (128
children, 71%) or the intensive care unit (13 children, 7%).
Viral testing Thirty-nine (22%) children were evaluated in the emergency
Rapid viral testing was conducted for clinical care by us- department and discharged home. On average, children had
ing an indirect fluorescent-antibody (FA) assay to test for respiratory symptoms for 4.8 days prior to sample collection.
RSV, PIV types 1–4, adenovirus and influenza A and B) Thirty-seven (21%) children had at least one of the follow-
(13). FA assays were not available for human metapneu- ing chronic disease conditions: asthma (n = 24; 13%), car-
movirus (hMPV) or coronavirus for this study. Patient res- diac condition (n = 12; 7%), pulmonary condition (n = 4;
piratory samples were later analysed by multiplex real-time 2%), renal condition (n = 2; 1%), malignancy (n = 2; 1%),
PCR without prior knowledge of the FA result for the follow- gastrointestinal condition (n = 1; 0.6%) and neurological
ing viruses: RSV, adenovirus, hMPV, coronavirus, PIV types condition (n = 1; 0.6%).
1–3, and influenza. PCR was performed on total nucleic Real-time PCR and FA detection methods detected one
acids isolated from frozen nasal wash samples using a previ- or more respiratory viruses in 168 (93%) of the 180 pa-
ously described one-step reverse-transcription PCR master tients. A single virus was detected in 127 (71%) children
mix for RNA viruses and a master mix for adenovirus DNA and multiple viruses were detected in 41 (23%) children.
(13–15). Primer and probe sequences were designed us- RSV was detected in 139 (77%) children, adenovirus in 27
ing Primer Express Software (Applied Biosystems, Fos- (15%), hMPV in 19 (11%), coronavirus in 14 (8%), PIV in 10
ter City, CA, USA) as previously described (13–15). Prior (6%), and influenza in 1 (1%) (Table 1). Of the 41 children
studies indicate that the FA and real-time PCR viral with multiple viruses detected, RSV was the most common
testing was strongly correlated for both RSV and in- pathogen followed by adenovirus and coronavirus (Table 2).
fluenza. PCR was more sensitive than FA for PIV and A virus other than RSV, either alone or in combination with
adenovirus (13). another virus, was detected in 63 (35%) of children.

Table 2 Detected dual infections among viral respiratory pathogens as identified by FA and/or real-time PCR in 40 children with bronchiolitis

Age RSV+ Adenovirus RSV + Coronavirus RSV+ hMPV RSV + PIV RSV + Influenza A hMPV+ PIV hMPV + Adenovirus PIV + Adenovirus
n (%)1 n (%) n (%) n (%) n (%) n (%) n (%) n (%)

<6 months (n = 7) 1 (14) 3 (43) 3 (43) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
≥6 to 8 (50) 3 (19) 0 (0) 0 (0) 0 (0) 3 (19) 1 (6) 1 (6)
<12 months (n = 16)
≥12 years to 8 (47) 5 (29) 0 (0) 1 (6) 1 (6) 0 (0) 2 (13) 0 (0)
<24 months (n = 17)
All ages n = 40 17 (43) 11 (28) 3 (8) 1 (5) 1 (3) 3 (8) 3 (8) 1 (3)

b
Percentages are reflective of pathogens detected within a specific age group.

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Stempel et al. Viral pathogens in bronchiolitis

Of the 139 children with RSV detected by FA and/or real- between RSV and RV (11). In our study, we identified co-
time PCR, 34 children (24%) had one or more additional infections in 41 (23%) children with bronchiolitis despite
pathogens identified. RSV co-infections occurred most fre- not testing for RV. In contrast to the studies by Ong and
quently with adenovirus (17 samples, 50%), followed by Greensill, our study population was larger, encompassed an
coronavirus (11 samples, 32%), hMPV (3 samples, 9%), PIV entire respiratory season, and comprised patients discharged
(1 sample, 3%) and influenza (1 sample, 3%). One speci- from the emergency department or admitted to a general pe-
men contained three respiratory viruses (RSV, hMPV, and diatric floor or the intensive care unit.
adenovirus) and was contributed by a 3 month old evalu- The AAP Guidelines recommend limiting laboratory tests
ated in the emergency department for respiratory distress. for disease diagnosis but state that virological testing may be
There were seven co-infections that occurred in the absence beneficial if patient cohorting is feasible (3). Many facilities
of RSV (Table 2). use FA testing to direct cohorting, and Hall and Lieberthal
emphasize that the rapid tests available are typically limited
DISCUSSION to RSV and influenza and perform with variable reliabil-
We demonstrated a high rate of respiratory virus detection ity (17). Using more sensitive assays, we found that of the
using sensitive molecular-based assays among a large sample 139 children with RSV detected, 34 (24%) were infected
of children evaluated for bronchiolitis in a hospital setting. with another pathogen and most of these pathogens are not
We detected at least one virus in 93% of children with clini- detected well or at all with current FA assays. Interpreting
cal a diagnosis of bronchiolitis. RSV was the most common copathogen data with organisms such as coronavirus or ade-
pathogen but was detected in only 77% of children. A virus novirus is problematic due to the difficulty in differentiating
other than RSV was detected in 35% of children, and co- acute disease from long-term shedding and the extent that
infections occurred in 23% of children. bronchiolitis may be caused or exacerbated by these other
The development of sensitive molecular diagnostic assays viruses has not been definitively established. Our results sug-
has increased the number of viruses detected in compari- gest that the issue of viral testing merits further investigation
son to conventional methods. A previous study analysing as it has the potential to impact patient cohorting and man-
the viral detection capacities of culture, direct immunoflu- agement algorithms (18).
orescence, and multiplex PCR in children with acute respi- Limitations of our study include the retrospective design
ratory illness found PCR to be the most sensitive method and the use of residual clinical samples. In addition, we did
with 91.5% of children having a virus detected in their nasal not extend the study into the summer months, potentially
samples (7). Another study in infants with acute lower res- decreasing the detection of parainfluenza virus infections.
piratory tract infections performed PCR on nasopharyngeal Although we tested for a wide spectrum of viruses, viral de-
aspirates and detected viruses in 77% of patients (8). These tection of bocavirus and picornaviruses, such as rhinovirus,
studies found RSV to be the first (43.6%) or second (28%) were not included in this study due to limitations of spec-
most commonly detected virus, with rhinovirus (RV) being imen volume. Despite this limitation, we identified one or
the second (31.8%) or first (32%) most frequent virus, re- more viruses in 93% of samples.
spectively (7,8). Our study demonstrated a similarly high Results of our study not only confirm previous obser-
frequency of RSV, followed by adenovirus and hMPV, but vations that RSV is the most frequently detected virus in
we did not test for RV. patients with bronchiolitis, but also highlight the potential
The use of molecular techniques for viral detection has significance of other viral pathogens such as hMPV and ade-
increased the identification of multiple viruses in a single novirus in this clinical setting. Given the substantial pres-
sample (13). The prevalence of co-infections in other stud- ence of viral co-infections in RSV bronchiolitis, providers
ies has ranged from 19–35% in young children with diverse need to recognize the potential limitations of rapid RSV test-
types of respiratory tract infections seen in the hospital ing alone for cohorting purposes. Our data underscore that
or emergency department (7,8,12). Of the published stud- further study is warranted to determine the clinical impact
ies that use molecular diagnostics to report co-infections in of viruses other than RSV for children with bronchiolitis.
children with bronchiolitis, few analyse children diagnosed
during an entire respiratory season. A study by Ong et al. of AKNOWLEDGEMENTS
50 infants with RSV bronchiolitis hospitalized during a one- The authors would like to thank Laurel Laux, Judson
month period identified dual infection in 5 (10%) patients Heugel, Anne Cent and the University of Washington Clini-
(9). A study by Greensill et al. of 30 infants with bronchi- cal Virology Laboratory for their contributions to this study.
olitis requiring mechanical ventilation reported a 70% co- Funding for this study was provided in part by National In-
infection rate among hMPV and RSV (16). A co-infection stitutes of Health, grant HL081595. Dr Zerr had full access
rate of 9% was reported by Mansbach et al. in a multicen- to all the data in the study and takes responsibility for the
tre study that sampled children with bronchiolitis for two integrity of the data and the accuracy of the data analysis.
to three-week periods (10). Only one previous study was
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