Since the detection of human bocavirus (HBoV) from respiratory tract samples from Swedish infants and children with lower respiratory tract disease,1 2–10 2,8 clinical characteristics of HBoV as a single infection in Spain. To determine whether HBoV infections have some singular characteristics, HBoV-single infections were compared with the most prevalent respiratory virus-single infections in the same population.
PATIENTS AND METHODS
This was a substudy of an ongoing prospective investigation of respiratory tract infections in children, funded by FIS (Fondo de Investigaciones Sanitarias–Spanish Health Research Fund) grants numbers 98/0310 and PI06/0532, and approved by The Medical Ethics Committee.
Clinical Assessment.
The study population were children, less than 14 years of age, with respiratory tract infection who were admitted to the secondary public hospital Severo Ochoa (Leganés, Madrid), between September 2005 and August 2007 (patients recruited between September 2005 and August 2006 were included in a previous study11
During the hospital stay, and as part of the study, a physician filled out a study-questionnaire with the following variables: age, sex, clinical diagnosis, history of prematurity and underlying chronic diseases, need for oxygen therapy assessed by transcutaneous oxygen saturation, axillary temperature ≥38°C, presence of infiltrate/atelectasis in radiographs, administration of antibiotic therapy, duration of hospital stay, total white blood cell count, C-reactive protein serum values, and result of blood culture when it was done. In this study, asthma—or recurrent wheezing—was not considered a chronic underlying disease. Oxygen therapy was provided to achieve oxygen saturation ≥94%.
Upper respiratory tract infection was defined as the presence of rhinorrhea and/or cough in the absence of wheezing, dyspnea, crackling rales, or bronchodilator use, with or without fever. The classic criteria, present in an initial episode of acute onset expiratory dyspnea with previous signs of viral respiratory infection—whether or not this was associated to respiratory distress or pneumonia—were applied in diagnosing bronchiolitis.12
Virus Detection.
Specimens from patients consisted of nasopharyngeal aspirates (NPA) taken from each patient at admission (Monday through Friday). Each specimen (1 for each patient) was sent for virologic investigation to the Influenza and Respiratory Virus Laboratory at the National Microbiology Center (ISCIII, Madrid, Spain). Specimens were processed within 24 hours after collection. Upon receipt of NPA, 300 μL samples were aliquoted in the sample reception unit, where a total of 3 aliquots were prepared and stored at −70°C. The reception area and the NPA sample aliquoting area were separate from the working areas.
Nucleic Acid Extraction.
Total nucleic acids from 200 μL aliquot of clinical specimens were automatically extracted using the BioRobot M48 workstation and the MagAttract Virus Mini M48 Kit (Qiagen, Hilden, Germany) for polymerase chain reaction (PCR) assays. A cloned amplified product of a DNA fragment supplied by the Promega Access RT-PCR system kit (Promega) was obtained and added to the lysis buffer as an internal control for checking both the efficiency of acid nucleic extraction and presence of amplification inhibitors. Internal control was quantified in such a way a total of 100 molecules per tube were included.
PCR Methods for Detection of HBoV and 15 Other Respiratory Viruses.
Nucleic acids extracted from all NPAs were tested for HBoV using a sensitive and specific nested PCR method described previously.11 13 14 15
Appropriate precautions were implemented to avoid false-positive results by carryover contamination. Positive results were confirmed by testing a second aliquot of sample.
Statistical Analysis.
Clinical characteristics of infections associated to HBoV were compared with those associated with RSV, adenovirus, and rhinovirus infections. Patients with multiple infections were excluded from the analysis. Values were expressed as percentages for discrete variables, or as mean and standard deviation for continuous variables. Clinical characteristics and laboratory variables were compared using Student t test, Mann-Whitney U test, χ2 test, and Fisher exact test. A 2-sided value of P < 0.05 was considered statistically significant. All analyses were performed with the Statistical Package for the Social Sciences (SPSS), Version 13.0.
RESULTS
Screening of Virus and Patient Characteristics.
The study population consisted of 806 hospitalized children younger than 14 years of age, with acute respiratory infection. Since 86 patients were excluded either because of lack of NPA clinical samples or because they refused to participate, a total of 710 infectious episodes were analyzed. At least 1 respiratory virus was detected in 435 samples (61.2%), of which 308 samples corresponding to 308 episodes were single virus infections (70.8%), and 127 were dual or multiple viral infections (29.2%). Excluding coinfections, all viruses detected are listed in Table 1 in descending order of frequency. HBoV was detected in 99 patients (13.9%) corresponding to 35 single virus infections (35.4%) and 64 multiple virus infections (64.6%). All patients with single infections were younger than 7 years old.
TABLE 1:
Clinical Findings Associated With the Presence of HBoV, RSV, Rhinovirus, and Adenovirus.
Clinical characteristics of the 35 single virus bocavirus infections compared with the single infections associated to the most prevalent viruses detected, RSV (n = 131), rhinovirus (n = 66), and adenovirus (n = 40), are shown in Table 2 .
TABLE 2: Clinical Characteristics Associated With Infections Caused by HBoV Compared With RSV, Adenovirus (ADV), and Rhinovirus (RV)
HBoV Versus RSV.
Thirty-five patients with HBoV infection were significantly older (P = 0.017) than those with RSV infections (20.8 ± 25 versus 9.76 ± 13 months). Half of patients with HBoV were less than 12 months old (range between 1 and 147 months) and half of patients with RSV infection were less than 5 months old (ranged between 1 and 86 months). Table 3 shows age differences among groups. Bronchiolitis was observed more frequently in the RSV group (P = 0.0001) than in HBoV infected patients (Table 2 ). To rule out the confounding factor of younger age when diagnosing bronchiolitis in RSV infected children, the sample was stratified below and above the median age (297 days). For the group less than 297 days old, the statistical significance (P < 0.005) was maintained for the diagnosis of both viruses and bronchiolitis. Serum C-reactive protein and white blood cell count were higher in the HBoV group.
TABLE 3:
HBoV Versus Adenovirus.
The patients with adenovirus infections had higher fever (P = 0.036), and received antibiotics as treatment significantly more often (P = 0.009) than the HBoV group. Bronchiolitis was less frequently diagnosed in the adenovirus group (P = 0.017) and C-reactive protein had higher values in HBoV group (P = 0.043).
HBoV Versus Rhinovirus.
With exception of hypoxia, no significant clinical differences were found between HBoV and rhinovirus infections. Patients with rhinovirus infection required administration of oxygen less frequently than HBoV group (39.4% rhinovirus versus 62.9% HBoV, P = 0.0025).
DISCUSSION
According to our results, HBoV circulates in Spain with a very high incidence. In our study population (hospitalized children), we were able to identify HBoV in 99 children, yielding a frequency rate of 13.9%, almost one-third of which (35 of 99) was as a sole pathogen. HBoV ranked fourth of all viruses involved in single virus respiratory infections (Table 2 ).
In the original series described by Allander et al,1 2 3 5 4 10 6 8 16 3,7 16 2 11,17
A major strength of this study is the use of a panel of sensitive PCR assays,11,13,14 2 18,19
HBoV infections differ from other respiratory infections in several aspects and features. Our study indicates an association between HBoV infection and lower respiratory tract infections on the basis of its frequent detection in samples obtained from individuals hospitalized with acute respiratory illness. The most common reason for HBoV-infected children to be hospitalized was exacerbation of asthma followed by bronchiolitis. RSV is associated with bronchiolitis more frequently and the age of the patients is lower than in HBoV infected children. Leukocytes and RCP values are higher in HBoV infected patients. If we compare HBoV infections with those caused by adenovirus, bronchiolitis is less common in patients with infection caused by adenovirus. They have higher temperature and, perhaps for this reason, these patients received antibiotic therapy more frequently than the HBoV group. Infections caused by HBoV and rhinovirus have a similar profile in most of the analyzed features, perhaps because both cause asthma or recurrent wheezing in children of comparable age. However, children with infections caused by rhinovirus received oxygen therapy less often than those infected by HBoV.
Proof of causality is difficult to demonstrate and rarely emerges from 1 study, but depends on the accumulation of evidence.20 clinical characteristics in infections associated with HBoV compared with those caused by other respiratory viruses. This supports the idea that HBoV is a frequent cause of respiratory diseases in children. The absence or infrequency HBoV detection in healthy children also supports the pathogenic role of this virus in respiratory diseases in children. In the study of Kesebir et al7 21
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