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Undiagnosed Respiratory Viruses in Children

^a Division of Infectious Diseases, Department of Pediatrics, Center for AIDS Research, University of California, San Diego, California
^b Division of Infectious Diseases, Department of Pediatrics, Naval Medical Center, San Diego, California

	ABSTRACT

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OBJECTIVE. An estimated 12 to 32 million upper respiratory infections occur in young children each year. In addition, 20% to 53% of infants will have 1 episode of lower respiratory infection in the first year of life. The current methods of diagnosing respiratory viruses are limited in scope and sensitivity. Polymerase chain reaction is a more sensitive method than antigen detection and is often used for newly discovered viruses. Using polymerase chain reaction, we sought to diagnose adenoviruses, human bocavirus, and human metapneumovirus at our children's hospital.

METHODS. Nasal-swab specimens submitted for antigen detection of respiratory viruses were collected and processed by using polymerase chain reaction to detect adenoviruses, human bocavirus, and human metapneumovirus. Inpatient and emergency department records were reviewed for clinical and demographic data.

RESULTS. Approximately 1500 specimens were collected over 21 months; they contained adenoviruses, human bocavirus, and human metapneumovirus in 5.9%, 5.6%, and 5.2% of children, respectively. Using polymerase chain reaction and antigen detection, a viral agent was identified in as many as 62% of the specimens. Lower respiratory tract disease was present most frequently in patients infected with human metapneumovirus (63%) and least frequently in those infected with adenoviruses (45%). We detected adenoviruses by polymerase chain reaction in 59 patients for whom the antigen-detection test results were negative. A paroxysmal cough led to clinical suspicion of Bordetella pertussis infection in 19% of patients infected with human bocavirus.

CONCLUSIONS. Adenoviruses, human bocavirus, and human metapneumovirus were each present in 5% of specimens submitted for respiratory virus rapid testing. The lower respiratory tract was more commonly affected in patients with human bocavirus and human metapneumovirus infections. Adenovirus was often undiagnosed by antigen detection. Other findings included the presence of a pertussis-like illness associated with human bocavirus.

Key Words: human metapneumovirus • human bocavirus • adenovirus • PCR

Abbreviations: URI—upper respiratory infection • LRI—lower respiratory infection • RSV—respiratory syncytial virus • PIV—parainfluenza virus • HBoV—human bocavirus • DFA—direct fluorescent antigen • PCR—polymerase chain reaction

Although respiratory viral infections are generally uncomplicated, the cumulative impact of such illnesses is considerable. Upper respiratory infections (URIs) occur between 3 and 8 times a year in infants,¹ accounting for an estimated 12 to 32 million episodes of URI affecting the 4 million children born in the United States yearly.² URI can lead to acute asthma exacerbations, acute otitis media, and lower respiratory infections (LRIs).^3–5 The economic impact of URIs in the United States may be as high as $40 billion.⁶ In addition, it has been estimated that between 20% and 53% of infants will have 1 episode of LRI in the first year of life.^7,8 Hospitalizations for bronchiolitis have been estimated to cost (in 1998) as much as $300 million each year.⁹

Viral respiratory infections in children are commonly caused by rhinoviruses, respiratory syncytial virus (RSV), adenoviruses, influenza viruses, and parainfluenza viruses (PIVs), which are most frequently identified by culture or antigen detection.¹ Molecular laboratory techniques have led to the discovery of new respiratory viruses, such as human metapneumovirus¹⁰ and human bocavirus (HBoV).¹¹ In clinical practice, a specific agent is often not identified, although using molecular diagnostic techniques, a pathogen can be detected in 87% of children with acute respiratory infections.¹² At our institution, the sensitivity of the direct fluorescent antigen (DFA) detection for adenoviruses has been questioned on the basis of negative results by DFA in children with clinical disease consistent with adenoviral illnesses. Using the polymerase chain reaction (PCR), we sought to describe undiagnosed respiratory illnesses caused by 2 newly described viruses (metapneumovirus and HBoV) and to examine the sensitivity of the clinical diagnostic assay for adenovirus at a children's hospital.

	METHODS

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Specimen Collection and Processing
This study was a retrospective analysis of nasal scrapings submitted for diagnostic testing for respiratory viruses by DFA detection of RSV, influenza viruses A and B, PIVs, and adenoviruses using Light Diagnostics' respiratory virus panel I DFA (Millipore Corporation, Billerica, MA). For clinical purposes, specimens were collected at the provider's discretion. Specimens from patients between birth and 18 years were included, with no exclusions based on clinical characteristics of the patient. All of the specimens accepted for clinical testing (presence of cells was required) were included, regardless of results from DFA screening. Patients with multiple positive samples collected within a 1-month period were counted only once in the data analysis. Specimens were submitted from Rady Children's Hospital emergency department, inpatient facilities, and associated clinics. Each year, from November through March, the laboratory policy states that all of the specimens be tested by DFA for the presence of RSV first, with DFA for PIV, adenovirus, and influenza viruses being completed for all of the RSV-negative specimens. The complete panel was done for all of the specimens during the rest of the year. Specimens collected between February 1, 2004, and October 30, 2005, were stored at –70°C, and, after institutional review board approval, nucleic acid was extracted from 50 to 200 µL of sample using QIAamp MinElute virus spin kit (Qiagen, Hombrechtikon, Switzerland) and stored at –70°C. All of the pre-PCR processing was undertaken in a separate location from PCR and post-PCR analysis.

Polymerase Chain Reaction
Human Bocavirus
PCR for HBoV has been published previously.¹¹ Briefly, 2 primer sets were used with CybrGreen detection. The primer set targeting the NP-1¹¹ was able to consistently detect 17 copies per µL of plasmid DNA. All of the positive samples from the first primer set were subjected to a second round of PCR by using primers targeting the NS1 gene described by Sloots et al.¹³

Metapneumovirus
Metapneumovirus diagnosis was made by using the LightCycler PCR machine (Roche, Basel, Switzerland) and the FastStart Lightcycler HybProbe kit (Roche) according to the manufacturer's recommendations. Amplification was undertaken as described previously,¹⁴ except the Taqman probe was replaced with Lightcycler HybProbes by using the sequences (5'–3') GGTACAACAACTGCAGTGACACCTTCAT-FITC and LC-705-ATTGCAACAAGAAATAACACTGTTGTGTGG-Phos. We optimized the PCR by using a plasmid containing the metapneumovirus N gene that was kindly donated by Dr John Williams (Vanderbilt University, Nashville, TN) and were able to consistently detect 64 copies per µl of plasmid DNA. Each clinical sample was then tested individually by adding 2.5 µL of extracted nucleic acid to 7.5 µL of mastermix buffer. Final melting analysis was achieved with continuous monitoring of fluorescence from 45 to 90°C. A specimen was considered to be positive if a single melting peak was measurable between 52 and 65°C (single nucleotide variations are known to occur in the probe region, resulting in the variable melting temperature range).

Adenovirus
Adenovirus diagnosis was made via Taqman probes by using the LightCycler and the FastStart Lightcycler HybProbe kit (Roche) as described previously.¹⁵ Modifications in the cycling parameters were as follows: preincubation, 95°C for 10 minutes (1 cycle); amplification, 95°C for 0 seconds, 55°C for 10 seconds, and 72°C for 12 seconds (45 cycles, with single fluorescence detection); and cooling, 40°C for 30 seconds. A sample was designated "positive" if a fluorescence curve with a crossing point at 40 cycles was clearly visible.

Patient Data Collection
Records from patients with samples containing a respiratory virus were reviewed for demographic data, clinical symptoms, laboratory results, radiographic findings, and the use of antimicrobial agents. All of the inpatient and Rady Children's Hospital emergency department records were also available for review. Records were not available for 43 patients, who were seen only in an outpatient clinic.

A probable bacterial pneumonia was defined as an abnormal pulmonary examination, fever, and a focal "infiltrate" or "consolidation" as determined by a pediatric radiologist. Radiographic findings consistent with bronchiolitis or viral lower respiratory tract infections included hyperexpansion, peribronchial thickening, or atelectasis. The clinician's initial diagnosis was taken from either the admission history and physical examination or the emergency department discharge paperwork.

Statistical Analysis
Statistical significance was defined as a P value of <.05. Comparisons were made among adenovirus, metapneumovirus, and HBoV data sets using the Fisher's exact test (3 variable) for categorical data and the Kruskal-Wallis test for continuous data.¹⁶

	RESULTS

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Screening Results and Distribution of Infected Patients
From a total of 1354 children, 1474, 1468, and 1470 specimens were tested for the presence of the HBoV, metapneumovirus, and adenovirus by PCR; results of 82 (5.6%), 76 (5.2%), and 87 (5.9%) were positive, respectively. All of the positive HBoV samples were also tested positive by confirmatory PCR. To establish the specificity of both primer sets, amplicons for both sets were sequenced from 10 patients found to have HBoV and all of the amplicons aligned with HBoV sequences in the gene bank. RSV was detected in 173 patients (12%). Coinfections are detailed in Table 1. The most common coinfections involved HBoV, with coinfections present in 22 (26%) of the 82 patients infected with HBoV. There were no coinfections with PIV or influenza viruses, which were present in 2.0% and 0.1%, respectively, of all samples.

View larger version (30K): [in this window] [in a new window]   FIGURE 1 Percent of positive specimens per month for RSV, metapneumovirus (MPV), HBoV, and adenovirus (ADV).

Clinical Descriptions
Records were available for review for all of the hospitalized patients, which included 71, 63, and 68 patients with adenovirus, metapneumovirus, and HBoV infections, respectively. The median age of infected patients was 1 year for all 3 of the groups (Table 2). The median lengths of stay (3–4 days) were similar for all of the groups. Patients with metapneumovirus were most often admitted to the ICU (19%) and had the longest oxygen requirement (median: 5 days).

The most notable differences on physical examinations of patients infected with adenovirus, metapneumovirus, and HBoV centered on abnormalities of the lower respiratory tract (Table 2), as defined by an abnormality in 2 of 3 of the following: hypoxia, increase work of breathing, and abnormalities on lung auscultation. Patients from whom adenovirus was identified using any method (PCR or DFA) had the lowest frequencies of hypoxia (21%), increased work of breathing (33%), and abnormalities on lung auscultation (40%) compared with patients infected with HBoV and metapneumovirus. Forty patients (63%) with metapneumovirus, 42 (61%) of those with HBoV, and 32 (45%) of those with adenovirus infections met the definition of clinical lower respiratory tract disease (P = .057). In addition, chest radiograph abnormalities consistent with bronchiolitis were most common in patients infected with HBoV (46%), followed by those with metapneumovirus (44%) and adenovirus (37%). Patients with radiographs interpreted as having an "infiltrate" or "pneumonia" were most commonly infected with HBoV (9.2%) and least commonly with adenovirus (3.7%).

Clinical Diagnoses and Antibiotic Use
Each diagnosis for a single patient was recorded when multiple discharge diagnoses were listed (Table 3). The most common diagnosis among all of the infections diagnosed by PCR was bronchiolitis, followed by uncomplicated URI, bacterial pneumonia, and asthma. Patients infected with adenovirus were frequently given diagnoses of suspected adenovirus, conjunctivitis, suspected Kawasaki disease, and fever without a source. Five patients (7.4%) from whom adenovirus was identified also had bacterial urinary tract infections. Thirteen patients (19%) infected with HBoV were suspected of having a pertussis infection (P = .001). Ranging between 7% and 10%, the frequency of acute otitis media was similar among patients infected with adenovirus, metapneumovirus, and HBoV. Although an infrequent event, the increased frequency of seizures occurring in children from whom metapneumovirus was isolated was statistically significant (P = .042).

	DISCUSSION

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Both clinicians and parents find satisfaction in being able to put a name to a child's illness, and, likewise, frustration in the words "it's probably just a virus." As molecular laboratory techniques become more widespread, we are closing the gap on undiagnosed respiratory illnesses. A recent example of the power of molecular methods of pathogen discovery is the discovery of human metapneumovirus using nonspecific priming, followed by a comparison of the sequenced nucleic acid with known pathogens.¹⁰ Metapneumovirus has been implicated in 20% of respiratory illnesses in children.¹⁷ Other recently discovered viruses included new coronaviruses^18–20 and HBoV,¹¹ accounting for an additional 8.8%¹⁸ and 5.7%²¹ of respiratory illnesses in children, respectively. Often thought to cause mainly URIs, the important role rhinoviruses in the lower respiratory tract has also been described recently.²² Using a variety of techniques to detect 7 different respiratory viruses, Jennings et al¹² were able to detect a pathogen in 87% of children with acute respiratory infections.

Although our study was not designed to determine the impact of PCR on clinical decision-making, we do demonstrate the large burden of viral disease missed by current clinical laboratory techniques. Adenoviruses, HBoVs, and metapneumoviruses were each present in 5% of our population over a 21-month period, but, more importantly, each pathogen was present at peak prevalence as high as 21% during different months. Adding the PCR detection for the very common pathogens, such as RSV, picornaviruses, and coronaviruses, would certainly increase our rate of pathogen detection, as has been shown previously.^12,22,23

Comparing the severity of LRI revealed that, in our population, adenovirus tended to cause less severe LRI than metapneumovirus or HBoV, although the differences seen were not statistically significant (Table 2). As seen in our study, adenovirus causes nearly 6% of respiratory tract infections in children and has been shown to be a leading cause of LRI in children.^24,25 Adenoviruses have been implicated in bronchiolitis obliterans pneumonia, as well as severe pneumonias mimicking bacterial pneumonia.^26,27 In our study group, patients infected with adenovirus less frequently had hypoxia, dyspnea, or abnormalities on lung auscultation and were more likely to have normal chest radiographs (Table 2). Although severe adenovirus pneumonias occurred rarely in our population, the trend was toward milder LRI compared with patients infected with HBoV and metapneumovirus. By contrast, patients with adenovirus were more likely to be assigned diagnoses not specifically associated with the respiratory tract, such as suspected adenoviral infections, suspected Kawasaki disease, and fever without a localizing source (Table 3).

The sensitivity of antigen-detection tests is variable. In a report comparing the sensitivity of DFA to culture for RSV, PIV, adenovirus, and influenza viruses, the adenovirus DFA was the least sensitive of the panel (47% sensitive).²⁸ Rocholl et al²⁹ examined the charts of 4568 children tested for adenovirus using antigen detection and found the sensitivity of their antigen-detection technique to be only 63% compared with culture. At our institution, adenovirus DFA was suspected to be insensitive, based on infrequent detection. We identified an additional 59 patients with adenovirus that had not been diagnosed by DFA testing, supporting the concept of superior sensitivity of PCR over antigen detection for the diagnosis of adenovirus.^23,30 We did not compare the sensitivity of the other viruses tested by DFA at our institution, although it is possible that influenza virus, PIV, and RSV infections may have been missed as well.

Several recent studies on HBoV have been published since its original description,^11,31 with ranges in prevalence from 1.5% to 19.0%.^{11,13,21,31–34} These studies show HBoV as a cause of URIs. In an important step toward proving causation as a true respiratory pathogen, Kesebir et al³⁴ found HBoV in 22 (5.2%) of 425 children with respiratory symptoms and in 0 of 96 asymptomatic children. The frequency of LRI symptoms in infected patients in our population, as well as in other recent reports,^31,34 suggests that it may also be a pathogen of the lower respiratory tract. In addition, we found that not only did patients infected with HBoV often have symptoms consistent with lower respiratory tract disease, they were also often suspected of being infected with Bordetella pertussis because of the presence of a paroxysmal cough and were frequently treated with a macrolide antibiotic for this reason.

In our study population, an additional virus was detected in 28% of the subjects who tested positive for HBoV, which was more frequent than for coinfections occurring with adenovirus (12%) and metapneumovirus (18%), although the difference was not statistically significant (P = .272). The high frequency of codetection of other respiratory viruses with HBoV has been described by others, as well,^22,31,35 although the clinical significance of this trend is unknown. Because the presence of viral nucleic acid in the upper airway in children with lower respiratory tract disease does not establish HBoV as a causative agent of LRI, detection of HBoV from lower respiratory tract samples will be important to verify its role in LRI.

	CONCLUSIONS

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The conclusions of this study are limited by its retrospective nature. Because the specimen collection was based on the providers' discretion, the symptoms described are biased toward the general indications for viral testing by DFA. Many children would have been tested because of the presence of respiratory symptoms, although indications such as seizures and fever without source would have lacked such symptoms. The frequency of hospitalization and severity of symptoms caused by adenovirus, metapneumovirus, and HBoV have likely been exaggerated, because viral diagnosis would be sought more frequently for children requiring hospitalization than those treated as outpatients with less severe disease.

These data suggest that, at our institution, there is a large burden of undiagnosed viral illnesses caused by HBoV, metapneumovirus, and adenovirus. LRIs tended to be more commonly caused by metapneumovirus and HBoV, whereas adenovirus infections had a more protean spectrum of presentation. The diagnosis of adenovirus was frequently missed by antigen-detection techniques as compared with PCR. Whether comprehensive and sensitive viral diagnostics make a clinical impact worth the extra time and expense is still unclear.

	ACKNOWLEDGMENTS

 
This work was supported by in part by grant AI-36214 (Virology Core, University of California, San Diego, Center for AIDS Research).

We thank Teresa Mueller and Charles (Chuck) Woo for constant help in the rapid diagnostics laboratory at Rady Children's Hospital. In addition, we thank the Department of Pathology staff at Rady Children's Hospital, particularly Dr Denise Malicki, without whose support this study would not have been possible. We thank Dr Robert Riffenburgh, who was invaluable in the statistical analysis of our data. Finally, we acknowledge the support of Dr Kevin Russell at Naval Health Research Center in San Diego, who provided the plasmid for human adenovirus, with which our PCR assay was optimized.

	FOOTNOTES

 
Accepted Aug 16, 2007.

Address correspondence to John C. Arnold, MD, Department of Pediatrics, Division of Infectious Diseases, Naval Medical Center, San Diego, 34800 Bob Wilson Dr, San Diego, CA 92134. E-mail: john.arnold{at}med.navy.mil or Mark H. Sawyer, MD, Children's Hospital and Health Center, 3020 Children's Way, MC 5041, San Diego, CA 92123. E-mail: mhsawyer{at}ucsd.edu

The authors have indicated they have no financial relationships relevant to this article to disclose.

The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, or the US Government.

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This Article Abstract Full Text (PDF) P3Rs: Submit a response Alert me when this article is cited Alert me when P3Rs are posted Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Arnold, J. C. Articles by Sawyer, M. H. PubMed PubMed Citation Articles by Arnold, J. C. Articles by Sawyer, M. H. Related Collections Infectious Disease & Immunity

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