Conclusion: HBoV1 was detected by PCR in one fifth of the children with non-severe CAP and acute HBoV1 infection was serologically confirmed in one quarter of these cases. The real role of respiratory viruses in the pathogenesis of CAP remains debatable. This concerns especially human bocavirus 1 (HBoV1) which is mostly detected with other respiratory viruses and is often detected in asymptomatic subjects. 4 HBoV1 has, since its discovery in 2005, 5 been found in 10-20% of nasopharyngeal aspirate samples (NPA) of hospitalized children with lower respiratory illness. [6] [7] [8] However, HBoV1-DNA has been shown to persist for weeks or even months after an acute infection, detectable as low viral loads in the NPAs, questioning the clinical value of PCR-based diagnosis. 9 In 2008, it was shown that by measurement of HBoV1specific IgM and IgG antibodies, it was possible to diagnose acute HBoV1 infection in children hospitalized with acute severe respiratory diseases. 9 In the current study, we used specific serology to assess the frequency of acute HBoV1 infections in children with both non-severe CAP and with HBoV1-DNA detected by PCR in the nasopharyngeal mucus. The assessment of respiratory virus detection was a secondary objective in the clinical trial.
On enrolment, after written informed consent had been obtained from parents/legal guardians, every patient was evaluated by trained medical students under the supervision of a senior pediatrician, all from the research team. A complete physical examination was performed.
Data on demographics, clinical history, and physical examination were recorded in pre-defined forms with the following variables: age, sex, disease duration, complaints (cough, fever, difficulty breathing, wheezing, vomiting), axillary temperature, respiratory rate (RR), weight, findings on physical examination (chest retraction, reduced pulmonary expansion, rhonchi, wheezing, crackles). A follow-up visit was scheduled to occur 2-4 weeks after recruitment. Fever was defined as axillary temperature >37.4°C 12 and tachypnea as RR ≥ 50 breaths/min in children aged 2-11 months or RR ≥40 breaths/min in children from 12 to 59 months of age. 13 Nutritional evaluation was performed using the software Anthro, version 1.02 (Centre for Disease Control and Prevention and WHO) and malnutrition and severe malnutrition were defined as Z-score for weight-for-age index under −2.00 or −3.00, respectively, using the National Centre for Health Statistics standard. 14 In addition, an in-house PCR test was used to detect EV and RV in NPA samples, as previously described. 15 Due to the known long persistence 17 Borderline results in all EIAs were considered indeterminate.
The PCR methodology is highly vulnerable to possible false positive results due to contamination of reagents and specimens with PCR amplicons. The analyses were performed in an accredited clinical virus diagnostic laboratory. In the present work, rigorous measures were taken throughout the whole study to avoid amplicon as well as sampleto-sample contamination.
Sample size was estimated as 73 cases with HBoV1 detected in NPA, considering an expected frequency of acute serologically diagnosed HBoV1 infection equal to 5%, total width of confidence interval (CI) of 10% and 95% CI. We performed a bivariate analysis: the frequency of each clinical characteristic of children with or without acute HBoV1 infection was compared by using chi-square or Fisher's exact test as appropriate; continuous variables were presented as median (interquartile range [IQR]) and were assessed by using Mann-Whitney U test due to non-parametrical distribution. The tests were two-tailed with significance at <0.05. As the frequency of missing information was very low, we chose to handle the absent data by excluding these cases. All laboratory procedures were performed by trained personnel blinded to clinical data. All analyses were performed using the Statistical Package for the Social Sciences (SPSS), Version 9.0. 
A total of 820 children were enrolled in the clinical trial. Of these, 759 (92.6%) children had NPA and acute and convalescent serum samples collected and were included in this study. The median (IQR) interval between collection of acute and convalescent samples was 19 (16) (17) (18) (19) (20) (21) (22) days. The widely used multiplex PCR tests have greatly increased our understanding of the role of respiratory viruses in pneumonia. In many studies in children with CAP, respiratory viruses have been detected in 50-70% of the cases. 4 However, many viruses including HBoV1 have been detected at similar or higher rates in asymptomatic controls which has questioned whether these viruses are causative agents of CAP. [21] [22] [23] This concept is debatable, because respiratory viruses may also cause asymptomatic infections which explains the detection of viruses in asymptomatic subjects. 15 Our study has certain limitations. The study was a single center study limiting wider generalization of the observations. Due to economical restrictions, HBoV1 serology was studied only from HBoV1-DNA-positive patients. It is probable that also studying HBoV1-DNA-negative CAP children would have increased our diagnostic yield. In the study on acute wheezing 13% of children 
Monthly distribution (n) of cases with acute human bocavirus 1 infection diagnosed by serology, of cases with human bocavirus 1 detected in nasopharyngeal aspirate samples, and of the recruited community-acquired pneumonia cases in each month of the study period who tested negative for HBoV1-DNA in the nasopharynx showed serologic evidence of acute HBoV1 infection. 9 Furthermore, we did not study HBoV1 copy numbers nor HBoV1 DNA in the serum.
However, it has been previously shown in a Finnish study that 38% of the children with low-load PCR positivity have acute infection diagnosed by serology whereas 96% of the high-load positive PCR have the same serological diagnosis. 16 Therefore, serology should be enough to show reliably the actual acute infections. Moreover, it was very recently published that when compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). 32 An additional limitation is that no virus-bacterial co-infections were analyzed. The strength of our study includes a long recruitment period lasting almost four and half years, reducing the risk of bias regarding viral seasonality, and a high number of CAP patients. In addition, the serologic studies were carried out in a well-established laboratory of HBoV1 virology. It has been recognized that serology is the most accurate method to diagnose acute HBoV1 infection, even when it was compared to detection of HBoV1 messenger RNA which implies viral replication. 32 In conclusion, our observations provide further support for the causal role of HBoV1 in the pathogenesis of childhood CAP, both as a sole agent or with other respiratory viruses.