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Little is known about HBoV persistence and shedding and the association between HBoV detection and the onset and resolution of respiratory symptoms.
We performed HBoV testing on nasal swabs from a prospective, longitudinal study of respiratory illness in 119 children attending daycare.
HBoV was detected in 70 children (59%), and in 106 (33%) of the 318 study illnesses. Another virus was detected in 76 HBoV+ illnesses (72%). Extended and intermittent shedding was observed, with consistent HBoV detection documented for up to 75 days. HBoV was detected in 20 of 45 asymptomatic enrollment samples (44%) and HBoV prevalence and viral load did not differ significantly between children with and without symptoms at enrollment. HBoV+ illnesses were longer than HBoV- illnesses (OR: 2.44 for symptoms > 7 days, 95% C.I. 1.41, 4.22) and illnesses with HBoV viral load ≥ 4 log-copies/mL required a visit to a health care provider more often than HBoV-illnesses (OR: 1.64, 95% C.I.: 1.02, 2.64).
HBoV was more common in illnesses with greater severity. However, detection of HBoV was not associated with the presence of respiratory illness or with specific respiratory symptoms in this prospective study of infants and toddlers attending center-based daycare.
Respiratory infections are the leading infectious causes of death worldwide. In addition to the clinical morbidity they cause, respiratory illnesses in children have been implicated in increased school absenteeism and parental work absenteeism.[2, 3] New viruses that potentially play a role in infectious respiratory illness are increasingly being recognized through sensitive methods of viral detection.
Since the initial detection of human bocavirus (HBoV) in 2005, numerous studies have been published documenting the detection of HBoV in nasal samples. Seroprevalence studies have also shown that HBoV is widespread among young children. However, most studies of the role of HBoV in respiratory illness are cross-sectional in nature or focus on moderate to severe illness, and more specific evidence is needed to define the role that HBoV plays in respiratory illness.[6, 7] Current information has not proven that HBoV is a respiratory pathogen rather than a colonizer. Little is known about the temporal relationship between HBoV detection and the onset and resolution of illness, and the relationship of viral load to disease severity is unclear. Few studies have used regular, repeated, longitudinal sampling; one longitudinal study in a birth cohort of Danish children found frequent detection of HBoV in both symptomatic and asymptomatic infants. To address these questions, we have performed HBoV testing on samples from a prospective, longitudinal study of respiratory illness in young children attending daycare.
Nasal swab samples and symptom data were collected as part of a longitudinal study of respiratory illness at 3 large daycare centers on Fort Lewis Army Base in Washington State.
Parents of daycare enrollees were recruited through flyers at the beginning of the study and when new children entered the daycare. Inclusion criteria were (1) ages 6 weeks through 24 months at enrollment and (2) attending at least 20 hours of daycare at one of the Fort Lewis daycare centers between February 2006 through February 2008. Informed consent was obtained in a consent conference with the on-site study nurse before enrollment. Children were enrolled prior to the initiation of respiratory virus surveillance and regardless of whether or not the child was symptomatic at the time of enrollment
The age distribution of children in the study varied by daycare classroom. Individual classroom age ranges included 6 weeks-12 months, 12-24 months and 24-36 months at Site 1, 6 weeks-24 months, 18-36 months and 24-42 months at Site 2, and 6 weeks-12 months and 12-24 months at Site 3 with 9-18 children per classroom. The percentage of children in each classroom enrolled in the study averaged 56% (range 35-71%).
After obtaining informed consent from the parents, nasal swabs were collected from children at enrollment (for children enrolled after May 1, 2006) and with each new respiratory illness for up to two years. Respiratory surveillance was conducted from February 2006 through April 2008. A new respiratory illness was defined as the onset of at least 2 of 5 symptoms: cough, rhinorrhea, wheezing, fever, nasal congestion. Study staff was notified of new or worsening respiratory illness by parents or by daycare staff. Active surveillance for respiratory illness was also conducted by the on-site study nurse.
At the onset of a new or worsened illness, data on the presence and duration of illness symptoms were collected by parent interviews performed by the study nurse and by daily symptom diaries completed by the parent for 10 days. Symptom data beyond day 10 was not collected. Medical record review and physician surveys were performed to collect data on health care visits related to study illnesses.
Nasal swabs were collected by deep nasal swab using flocked-tip Copan swabs, which were immediately submerged into a vial containing 0.5 mL lysis buffer and then stored at room temperature. Samples were tested during the study using nucleic acid extraction and PCR for 14 common respiratory viruses: respiratory syncytial virus (RSV) A and B subtypes, human metapneumovirus (HMPV), influenza (Flu) A and B, parainfluenza (PIV) types 1, 2, 3, and 4, adenovirus (AdV), rhinovirus (HRV), and coronavirus (CoV; subtypes 229E, HKU1, NL63, OC43),[9-13] with results available to study staff within one week. Repeat samples were collected weekly during respiratory illnesses until the child was negative for all viruses in the respiratory virus panel and their illness was beginning to resolve.
Study samples were tested retrospectively for HBoV using a real-time PCR assay targeting the NP1 gene  that provided semi-quantitative cycle threshold results only. HBoV+ specimens with sufficient volume (n=120) were tested again and specimen PCR threshold cycle values were compared to those of a standard curve generated by amplifying known copy numbers of a plasmid containing the HBoV amplicon. Quantitative results are reported as copies of virus per mL of lysis buffer. One-fifth of specimens positive by the qualitative assay had no HBoV detected by the quantitative assay. Cycle thresholds of these samples ranged from 36.4 to 39.7 in the initial assay, indicating that HBoV copy number in these samples was low.
Data was analyzed using STATA 10.1 (College Station, Texas). Incidence and 95% jackknife confidence interval of HBoV illness was calculated overall and individually for age and gender subgroups. Cox Regression models were used to determine independent risk factors for HBoV illness. An indicator term was included to adjust for respiratory season and robust standard errors were used to account for multiple HBoV illnesses per child. Children were included in this model beginning at the time of their first HBoV- sample.
Differences in the presence and duration of symptoms between illnesses with detectable HBoV and illnesses without were evaluated using generalized estimating equations with a robust estimator to appropriately account for the association between measures on the same subject.  Only illnesses with a positive HBoV detection in the first two illness samples (approximately one week of illness) were considered HBoV+ to minimize length bias. In response to a number of low-copy HBoV+ samples that were detectable by only one of two PCR assays, a sensitivity analysis was performed that required a quantitative value of at least 4 log-copies/mL to determine positivity. This cutoff was determined by rounding the 25th quartile quantity in our results to the nearest log. A corresponding cutoff of a cycle threshold value of 35.3 was used for samples with insufficient volume for quantitative testing; this cutoff value was selected because it linearly corresponds to 4 log-copies/mL based on our data. As a secondary analysis, characteristics of HBoV+ illnesses were compared to the characteristics of HBoV-illnesses immediately preceding and following the case event in each individual using a bidirectional case-crossover analysis design. This comparison was analyzed with generalized estimating equations with robust variance. Since this analysis required both an HBoV+ illness and a separate HBoV- illness for the inclusion of each child, the available dataset was markedly smaller than the full study analysis described above.
HBoV results for baseline enrollment swabs were compared between children with respiratory symptoms and children without using a χ2 test for differences in prevalence and a t-test for differences in log viral load. HBoV detection in asymptomatic children was also compared to the next consecutive illness sample in each child using McNemar’s test.
One hundred nineteen subjects were enrolled in the study, with a total follow-up time of 115.3 child-years and a mean follow-up of one year (range: 11 days to 2 years). Average age at enrollment was 10 months (range: 1.6 to 24.9 months) and 56 (47%) of enrollees were girls (TABLE 1).
318 illnesses in 93 children were reported during illness surveillance, including 45 illnesses reported at enrollment. The overall annual incidence of respiratory tract illnesses was 2.8 per child-year (95% C.I. 2.3 to 3.4). Annualized incidence in individual children ranged from 0 to 30 respiratory tract illnesses per year. A total of 495 weekly samples were collected during the 318 illnesses and daily symptom diaries were available from 313 illnesses. Illnesses with missing diaries were excluded from analyses of symptom duration. Forty-one additional swabs were collected from children who were asymptomatic at the time of enrollment (48% of 86 total enrollment swabs). A total of 536 total nasal swabs were available for retrospective HBoV testing.
152 samples (28%) tested positive for HBoV, and the virus was detected in 106 (33%) of 318 study illnesses. 70 of 119 enrolled children (59%) had a HBoV+ sample during the study, although 13 of these children had HBoV detected only during an asymptomatic enrollment sample. Among illnesses with any virus detected, HBoV was detected significantly more often than any of the 14 respiratory viruses but rhinovirus (FIGURE 1). This was also true among illnesses with fever and illnesses requiring a health care visit.
Quantitative HBoV results were available for 95 HBoV+ specimens. Median HBoV viral load was 15,500 copies per mL (range 1160 to 1.8×1010; IQR: 5210 to 408,000). No relationship was identified between viral load and age at illness (p=0.941). HBoV+ specimens with other viruses identified had a lower median log viral load (3.9; n=47) compared to specimens with only HBoV detected (4.5; n=48) (p=0.04, Mann-Whitney test). However, the comparison of the maximum HBoV log viral load between illnesses with HBoV plus other viruses versus illnesses with only HBoV was not significant (mean log difference: -0.2; p=0.602, GEE).
Another virus was detected in 76 out of the 106 HBoV positive illnesses (72%). This rate was significantly higher than the percent of HBoV- illnesses with multiple viruses (24%; p<0.001). The number of coinfecting viruses in addition to HBoV ranged from one (n=40) up to 4 (n=2) (2 viruses: n=23; 3 viruses: n=11). HRV and AdV were the most common coinfecting viruses (n=37 and n=33, respectively) (FIGURE 1). However, the rate of HRV-specific coinfection in HBoV+ illnesses was significantly lower than in HBoV- illnesses (33% versus 54%; p=0.003).
The beginning and duration of HBoV detections did not consistently correspond with the timing of respiratory illnesses. Of the 106 HBoV+ respiratory illnesses, HBoV was not identified until the second weekly swab (second week of illness) or later in 22 (21%) cases. 32 of 70 children with HBoV+ detections had HBoV detected at more than one sample. Extended shedding with swabs at most one month apart was observed for up to 75 days. Twenty HBoV+ shedding events with consecutive HBoV+ swabs spanned the onset and resolution of multiple respiratory illnesses. Some illustrative patterns of HBoV shedding are presented in FIGURE 2. Recurrent detection with HBoV was documented in 18 children with multiple HBoV+ events separated by at least one HBoV negative swab. Altogether, recurrent detection was seen two (n=13), three (n=3), or four (n=1) times in individual children.
The incidence of all HBoV+ illnesses was 93 illnesses per 100 child-years (95% C.I. 71, 123). Age and gender specific incidence rates (TABLE 2) were not significantly different after accounting for correlated illnesses among individuals. No significant difference in HBoV incidence was observed between daycare centers. The seasonal distribution of HBoV+ illnesses closely resembled the distribution of all respiratory illnesses during the study period (FIGURE 3).
HBoV detection did not correlate with the presence of respiratory illness symptoms. We detected HBoV in 44% of asymptomatic enrollment samples and found no significant difference in the prevalence of HBoV at enrollment between children with and without respiratory symptoms (TABLE 3). There was no significant difference in the mean HBoV viral load found in enrollment samples with and without respiratory symptoms (p=0.694; TABLE 3). These findings were consistent when restricting the analysis to HBoV-only infections. No association was found at enrollment between age and the detection or quantity of HBoV, whether or not symptoms were present. A case-crossover comparison of asymptomatic baseline samples with the next subsequent respiratory illness in each child (n=25 children) found no significant difference in HBoV prevalence between the asymptomatic samples and the illness samples (52% and 32%, respectively, p=0.059).
101 illnesses had HBoV detected in the first two illness samples (within approximately one week of illness onset). No association was found between HBoV detection and the presence of the following illness symptoms: fever, wheezing, cough, rhinorrhea, congestion, vomiting, diarrhea, decreased appetite or activity, earache, fatigue, myalgia, malaise, rash, or difficulty breathing. However, extended respiratory illness, defined as symptoms other than rhinorrhea lasting longer than 7 days, was more common in HBoV+ illnesses compared to HBoV- illnesses (OR: 2.44; 95% C.I.: 1.41, 4.22; p=0.001). Specifically, HBoV+ illnesses had higher odds of cough lasting >7 days (OR: 2.09; 95% C.I.: 1.29, 3.39; p=0.003). HBoV alone with no other coinfecting viruses was not associated with any outcomes (including duration of cough or illness and health care provider visits) when compared to HBoV- illnesses, however the number of HBoV infections with no other viruses was small (n=30). Our results were not affected by controlling for age. One child with HBoV was admitted to the hospital for severe respiratory illness, and this child was also infected with RSV, CoV, HRV and AdV during the course of the illness.
HBoV viral load did not correlate with severity of illness. In a sensitivity analysis requiring at least 4 log-copies/mL or a cycle threshold ≤35.3 to determine positivity, the results for all illness characteristics were similar to the previous analysis, with the exception of health care visits. HBoV+ illnesses with quantity above the cutoff (n=47) more often required a visit to a health care provider (OR: 1.64; 95% C.I.: 1.02, 2.64; p=0.042). We did not identify a continuous effect between increasing HBoV viral load and greater duration of cough or illness or increased health care provider visits.
No significant difference in reported symptoms was found when comparing HBoV+ (n=30) and HBoV- illnesses (n=37) that were matched by individual. Duration of illness was significantly longer in HBoV+ illnesses (mean difference 0.93 days; 95% C.I. 0.17,1.69; p=0.016) as was duration of cough (mean difference 1.20 days; 95% C.I. 0.06,2.34; p=0.038).
HBoV was the second most commonly detected virus in our prospective, longitudinal study of children in daycare and was detected in 33% of respiratory illnesses. The virus was frequently redetected in individual children over extended periods of time. Our study calls into question the role of HBoV as a respiratory pathogen, in light of the high frequency of codetection of other viruses, the detection of asymptomatic carriage, the lack of correlation with illness onset, and the lack of correlation of viral load with severity of illness.
Coinfections with respiratory viruses were present in 72% of HBoV+ illnesses. This estimate is in range with previous findings of up to 83%. The high percentage of HBoV detections that are concurrent with detections of known respiratory viruses calls into question the specificity of observed associations between HBoV and disease. We also found that HBoV detections did not have a distinct seasonal pattern when compared to the overall distribution of all respiratory illnesses over two years, in contrast to other reports.[8, 17-19] This pattern indicates that HBoV may be detected with a relatively consistent prevalence among respiratory illnesses caused by other viruses.
Our HBoV prevalence in asymptomatic samples was surprisingly high, and was very similar to a Canadian study that reported HBoV in 43 (43%) of 100 asymptomatic children. Von Linstow et al has also reported a HBoV rate in samples from asymptomatic children above that found in symptomatic children. We did not find a difference in HBoV prevalence between asymptomatic samples and the next illness sample within each child. This analysis design has the advantage of allowing for the control of confounding by individual factors such as age. Several studies have evaluated the association between HBoV and illness with comparisons to asymptomatic control groups and found a low prevalence of HBoV in children without respiratory illness symptoms.[16, 21-24] Our results contradict these findings.
HBoV detection did not consistently correspond with the onset of respiratory illness. One-fifth of HBoV+ illnesses had an HBoV- swab at illness onset. We identified HBoV infections that spanned the incidence and resolution of multiple respiratory illnesses. Several HBoV infections persisted for an extended period of time. We documented shedding for up to 75 days with regular sampling. This reflects reports of extended shedding for at least 2 months in 32% of children with HBoV+ respiratory illness who underwent monthly testing. We also described extended shedding events that spanned the onset and resolution of multiple consecutive respiratory illnesses. The persistence of HBoV shedding beyond the resolution of single respiratory illnesses illustrates the difficulty in correlating an isolated HBoV+ result with an incident illness in a cross-sectional study. Given that we have demonstrated that multiple HBoV events separated by HBoV- swabs are possible in individual children, it is possible that persistent HBoV detections reported here and elsewhere[8, 17, 25] may be due to repeated infections rather than a single extended period of shedding. More frequent sample collection is needed to investigate this fully.
Viral load was not significantly lower during asymptomatic periods compared to periods of respiratory illness, and we did not see increased illness severity with increased HBoV viral load. We found increased viral load among single HBoV infections among individual specimens, similar to other reports [17, 23] but this comparison was not statistically significant at the illness level. Other groups have been unable to establish a link between HBoV viral load and disease severity as well.[26-28]
While we did not identify any differences in reported symptoms between HBoV+ and HBoV- illnesses, HBoV did appear to contribute to the severity of respiratory illness. Illnesses with HBoV detection, alone or in combination with other viruses, were more likely to last >7 days and specifically were more likely to have cough present for >7 days. HBoV infections with a viral load of at least 10,000 copies/mL were also associated with increased visits to a health care provider. These findings indicate that HBoV may exacerbate respiratory illnesses caused by other pathogens, or that more severe illness may initiate HBoV shedding events.
The identification of HBoV shedding in the stool of children with vomiting and diarrhea  has led to speculation that HBoV may be a causative agent of enteric disease.  We did not find any association between HBoV detection and vomiting or diarrhea as reported by parental diaries and interviews. However, our study was designed for surveillance of respiratory diseases, and illnesses with vomiting or diarrhea without respiratory symptoms may have been missed.
Our rate of HBoV detection in respiratory illness is higher than other published estimates of 2.7% to 21.7%.[30, 31] In a more direct comparison to published cross-sectional prevalence estimates, HBoV prevalence in our study among first incident respiratory illnesses was 35%. Notably, over one-fifth of the HBoV+ illnesses did not have detectable HBoV until one week after illness onset. Studies that only collect a sample at illness onset may have missed these positive detections. To our knowledge, this study is the first longitudinal study to test for HBoV in a daycare setting. We evaluated all respiratory illnesses, including mild illnesses that are often missed by studies of hospital or primary care patients. It is possible that our high rate may be due to the very young age of our study participants (6 weeks to 24 months at enrollment). We do not know if this high prevalence is universally reflected in young children in other regions or not attending daycare.
Our reported duration of shedding may be an underestimate. HBoV testing was performed retrospectively, and weekly swabbing for respiratory viruses may have been discontinued before the resolution of the HBoV shedding. Secondly, testing began at the start of respiratory symptoms. We would have missed the onset of the HBoV infection if it preceded the start of illness. Our HBoV incidence may also be an underestimate if respiratory illnesses were missed during the surveillance by study staff and parents. However, we believe that very few illnesses were missed, due to the regular contact with the daycare providers and the on site study nurse who promptly identified illnesses. By focusing our study on the daycare population, we may have missed primary HBoV infections in very young children. The symptoms of primary HBoV infection, if any, are unknown and merit further study in young infants.
Few studies have longitudinally studied HBoV in children with mild illness symptoms.  To our knowledge, no longitudinal studies to date have utilized weekly sampling during respiratory illnesses and paired analysis to compare asymptomatic to symptomatic periods. HBoV was not independently associated with particular respiratory symptoms; however our findings indicate that HBoV shedding may increase the duration of respiratory symptoms caused by other pathogens. We also documented long periods of persistent HBoV shedding, independent of the onset and resolution of respiratory illness. Overall, detection of HBoV was not associated with the presence of respiratory illness or with specific respiratory symptoms in this prospective study of infants and toddlers attending center-based daycare.
Funding: This work was supported by Grant Number SYN104-05D from the MedImmune, Inc. Investigator Initiated Research Program (PI: Englund). A.W. also is supported by National Institutes of Health / National Institute of Allergy and Infectious Diseases grant number K24 AI107113.
Potential Conflicts of Interest. J.A.E. and M.P.F. have received research funding from MedImmune, Inc. J.A.E. has received research funding from sanofi pasteur and Novartis. E.T.M. and D.Z. have received research funding from Vioguard, L.L.C. A.M., A.W., and J.K.: no conflict.
Meeting Information: This data was presented in part at the Infectious Disease Society of America 46th Annual Meeting, October 2008, Washington, D.C., Abstract #V-4164a