PCR amplification was performed on the Applied Biosystems Gene amp 9700 PCR (Applied Biosystems, USA) with the following program: preheating at 50°C for 20 min; template denaturation at 95°C for 15 min; 34 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 2 min and kept at 4°C until ready tousle.

RVP FAST assay detects influenza A and B (INFA, INFB) including subtypes H1N1 (1977), H1N1pdm09, H3N2, respiratory syncytial virus A and B (RSVA, RSVB), enteroviruses including rhinoviruses (EV/Rhi), human parainfluenza viruses 1–4 (PIV1-4), human metapneumovirus (hMPV), adenovirus (ADV), human coronavirus NL63 (hCoV NL63), hCoV HKU1, hCoV 229E, hCoV OC43, and human bocavirus (hBoV). The assay was performed according to the manufacturer's instructions. Assay performance was controlled using bacteriophage lambda included in every run. The assay comprised two steps: a multiplex PCR amplification step and hybridization step.

Statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Categorical variables between the two study groups were compared using a chi-square test, and continuous variables were compared using a Mann-Whitney test. Factors identified as significantly different between the ADV and coinfection groups in a univariate analysis were assessed for association with coinfection through a multivariate analysis using a binary logistic regression test. A P value of <0.05 was considered as statistically significant.

On admission, a nasopharyngeal swab was collected from children complaining of respiratory symptoms or fever without a focus. Samples were sent, as soon as possible, to the Department of Laboratory Medicine where a multiplex PCR test for respiratory viruses was conducted. The commercially available AdvanSure™ RV real-time PCR kit (LG Life Sciences Ltd., Seoul, Korea) was used to detect ADV, influenza A and B viruses, RSV, parainfluenza virus, rhinovirus, coronavirus, human metapneumovirus, and human bocavirus. A positive PCR result for ADV confirmed an ADV infection. If the PCR result was positive for only ADV, the child was assigned to the ADV group, and if the PCR result was positive for two or more viruses including ADV, the child was assigned to the coinfection group.

Upper respiratory tract infection (URTI) included acute pharyngitis, pharyngoconjunctival fever, and acute otitis media, while lower respiratory tract infection (LRTI) included acute bronchiolitis, acute bronchitis, and pneumonia.

Absolute quantification of DNA by qPCR revealed that the HBoV viral load in HBoV-positive patients varied very broadly, ranged from <500 to 1×109 copies per mL of sample material, with median value of 9912 (figure 1). The median viral load which was found in patients with sole HBoV infection was not higher than those who had co-infection with other respiratory viruses (p = 0.2517). A recent review suggested if serum samples were not available for PCR, and then the probable next best option was the quantitative PCR with a cutoff of >104 copies/mL of respiratory tract secretions. Several previous studies reported that increased serum HBoV IgM and IgG had been found in most of patients who had high HBoV load (>104 copies/mL) in respiratory tract secretions, which was associated with respiratory symptoms,,. Therefore the HBoV-positive samples were divided into 2 non-overlapping populations: one group of 15 samples with a high viral load (>104 copies/mL) and one group of 16 samples with a low viral load (<104 copies/mL).

Longer duration of hospitalization and days of wheezing were found among children with high HBoV loads (table 5). Then we looked at the correlation between viral loads and duration of hospital stay and days of wheezing, we searched for additional evidence of etiology by studying whether the viral load of HBoV was unrelated or correlated to the days of hospitalization and presented wheezing. And we found that the days of wheezing showed a direct correlation with viral load (Correlation coefficient = 0.538. P = 0.003) (Figure 2).

Multiplex PCR testing detected 166 viruses in 158 of 432 patients (37%) (Table 1) of whom 60% were men, with an average age of 70 years, and 40% were women, average age 73 years. No influenza B virus was found in study samples. Two samples positive for influenza A virus by routine testing could not be confirmed by multiplex PCR.

Influenza A virus was the most common finding, detected in 96/158 (61%) of study patients. During the same period, clinicians ordered the triple viral test for influenza A and B viruses and respiratory syncytial virus in 308 patients. Of those, 126 (41%) were positive for influenza A virus, 1 (0.3%) for influenza B virus and 7 (2%) for respiratory syncytial virus. During the first three study weeks, only one clinically requested test was positive for influenza A virus compared with six in the study group. During the first four weeks of the influenza epidemic there were few clinically requested tests for influenza virus. This indicates that early in the influenza epidemic, awareness among clinicians that the flu season had started was low (Fig. 2). The routine triple viral test for influenza A and B viruses and respiratory syncytial virus available to clinicians was requested in only 56 of 107 (54%) patients who turned out positive for those viruses in the study analysis.

The Pathofinder multiplex PCR used for the first 129 patients of the study was positive for M. pneumoniae in five cases. Two of these patients also tested positive in the clinically available test. During the latter part of the study, when PCR for M. pneumoniae was not included in the multiplex PCR, there was one additional patient positive for M. pneumoniae by routine PCR testing.

By conventional culture from the nasopharynx culture there were 101 out of 432 (23.4%) patients positive for any bacteria (Table 2).

Part of the aliquot was used for routine detection of viral antigen using immunofluorescence (IF) detection of viral antigens for five respiratory viruses, viz. influenza viruses types A & B, respiratory syncytial virus (RSV), parainfluenza virus, and adenovirus. It was also cultured for virus isolation. The remaining aliquot was used for polymerase chain reaction (PCR) detection of rhinovirus, human metapneumonvirus, human coronavirus NL 63, OC43, 229E, HKU1, and bocavirus. [6, 8, 19, 21]

A total of 117 nasopharyngeal swab (NPS) samples were included in our study and tested by Alere i RSV and Altona RealStar RSV RT-PCR. In three samples, the Alere i RSV assay was invalid, as the lyophilized test reagents were not properly dispensed due to a handling failure. These samples were excluded from the analysis.

The 114 remaining NPS were collected from patients hospitalized due to lower respiratory tract infection (n = 79), upper respiratory tract infection (n = 12), or nonrespiratory diagnoses, including, e.g., febrile convulsions or gastroenteritis with concomitant respiratory tract infection (n = 14). In 9 patients, the admission diagnosis was not reported. The median age of the patients included in our analysis was 12 months (range, 2 weeks to 17.7 years).

Altona RealStar RSV RT-PCR was RSV positive in 43% (49/114) and RSV negative in 57% (65/114) of cases. RSV A and B was detected in 20 (41%) and 29 (59%) of RSV-positive samples, respectively. The mean threshold cycle (CT) value of RSV-positive samples was 22.7 (95% CI, 21.4 to 24.0), and the median CT value was 22.3 (range, 15.0 to 33.8). Mean CT values did not differ between RSV A- and RSV B-positive samples (RSV A, 23.2; RSV B, 22.5; P = 0.6).

The Alere i RSV test result was true positive in 49 out of 49 samples and true negative in 63 out of 65 samples. We did not undertake comprehensive analytical specificity testing but note that the 65 samples with a negative Altona RealStar RSV RT-PCR result included samples which had previously tested positive for influenza A (n = 10), influenza B (n = 6), parainfluenza (n = 3), human metapneumovirus (n = 9), coronavirus (n = 7), rhinovirus (n = 17), parechovirus (n = 1), and adenovirus (n = 1). Of the 2 samples with diverging test results, 1 test result was interpreted as false positive, whereas the other test result was invalid due to sample interference (Fig. 1). In both cases, we could not conduct confirmation testing due to limited sample volume. For the calculation of the Alere i RSV specificity, both samples with diverging test results were considered false positive. We thus report a conservative estimate of the Alere i RSV performance.

The combined RSV A and B test sensitivity for the Alere i RSV was 100% (95% CI, 89 to 100%). The specificity of the Alere i RSV test was 97% (95% CI, 89% to 100%) (Table 1).

The Alere i RSV test includes a 3-min step of preheating the lysis buffer, followed by the nicking enzyme amplification reaction with a maximum duration of 10 min. The assay identifies positive test results as soon as an amplification threshold is reached. In this study, the median duration of the nicking enzyme amplification reaction in RSV-positive samples was 1.8 min (range, 1.7 min to 3.9 min). Hence, in RSV-negative samples, the total duration of the test assay was 13 min (3 min of preheating plus 10 min of amplification), whereas it was only approximately 5 to 7 min in RSV-positive samples.

Positive test results were identified quicker in samples with high viral load (CT value of <25), although the absolute differences are small and probably of minor importance in routine care (Fig. 2).

At time of recruitment, baseline nasopharyngeal swabs were collected by trained staff and tested in the Beijing CDPC laboratory. Detailed demographic and clinical details for all participants were also collected. This included age, sex, smoking history, comorbidities, vaccination status, medications, use of personal protective equipment, performing high risk procedures, antivirals and results of laboratory tests.

At the end of the 4 weeks, another set of nasopharyngeal swabs was collected from all participants. A second (exit) survey was administered at the same time to participants to collect information about the development of any respiratory symptoms in the previous 4 weeks. Clinical respiratory illness (CRI) was defined as two or more respiratory symptoms (cough, nasal congestion, runny nose, sore throat or sneezes) or one respiratory symptom and a systemic symptom (chill, lethargy, loss of appetite, abdominal pain, muscle or joint aches).

The primary endpoints were:Laboratory-confirmed bacterial colonisation in symptomatic/non-symptomatic subjects. Multiplex PCR was used to test for Streptococcus pneumoniae, Legionella, Bordetella pertussis, chlamydia pneumoniae, Mycoplasma pneumoniae or Haemophilus influenzae type B (Seegen, Inc., Seoul, Korea).Laboratory-confirmed viral respiratory infection in symptomatic/non-symptomatic subjects, defined as detection of Adenoviruses, Human metapneumovirus, Coronaviruses 229E/NL63 and OC43/HKU1, Parainfluenzaviruses 1, 2 and 3, Influenza viruses A and B, Respiratory syncytial viruses A and B, or Rhinoviruses A/B by nucleic acid testing (NAT) using a commercial multiplex polymerase chain reaction (PCR) (Seegen, Inc., Seoul, Korea).

Following the discovery of WUPyV in Australia, the virus was detected in specimens from patients with respiratory tract disease on all continents suggesting a worldwide distribution [10,29–31]. So far, WUPyV-DNA was reported to be found in respiratory tract specimens (e.g. nasopharyngeal washes, tracheal secretion, BAL), serum, and faeces. The virus could not be detected in urine or from UV light-associated primary malignant lymphomas. Specimens from other malignant diseases have not been investigated. The use of tracheal secretion for diagnostics has been shown to lead to an underestimation of the rate of positive specimens compared to other respiratory materials for HBoV. This may be true for WUPyV, too.

The data available are mainly based on retrospective studies exclusively including symptomatic patients. The detection rate in respiratory samples from children with respiratory disease varies from 0.4% to 11.5%. The age of WUPyV infected patients ranged from a few weeks to 53 years, children <3 years of age were dominating. Infections were predominantly detected in late winter, spring, and early summer. High infection rates were reported for study populations preselected for lack of immunocompetence. HIV positive patients had detection rates of up to 35.7% in respiratory tract specimens and 8.3% in blood. The rates of co-infection with established respiratory viruses lay between 30.8% and 91.7%, commonly exceeding 50%. WUPyV was detectable in blood, possibly indicating its potential for systemic infections.

Le and co-workers presented evidence for viral persistence, Wattier et al. for nosocomial infections with WUPyV.

The real time protocols available allow the quantification of WUPyV. Quantification of viral loads in respiratory tract specimens revealed viral titers up to 1010 copies/ml, but low and medium viral loads were dominating. No correlation between viral load and the rate of co-infection or clinical diagnoses was observed.

Few studies included asymptomatic control groups [34,36,42–44]. The results were not concordant and reached from higher detection rates in the control group to higher detection rates among the group of patients with respiratory tract diseases.

Prospective studies have been published only recently. Van der Zalm and co-workers reported the detection of WUPyV in a cohort of 18 children. Their parents were contacted twice a week over a 6-months period (November to April) and asked for symptoms of respiratory tract disease in their children. Every two weeks respiratory tract specimens were collected, regardless of respiratory symptoms. 11.5% of the specimens of children with symptoms were WUPyV positive, but only 3.1% of specimens of healthy children, indicating at least an association of WUPyV with disease.

HBoV1 is frequently found in respiratory tract samples collected from hospitalized children with a peak age up to 24 months [24–26]. By PCR it appears as the third most common pathogen next to RSV and rhinovirus in young children presenting with acute bronchiolitis and wheezing [4, 27, 28]. Furthermore, serological studies have revealed that HBoV1 acute infections occur most often in early childhood, with HBoV1 IgG seroconverting at a median age of 1.9 years and reaching a seroprevalence of 80% by 6 years of age. Several clinical cases have shown severe or even life-threatening respiratory tract diseases due to HBoV1 infection in children [10, 15–19, 29]. However, it should be kept in mind that, due to HBoV1 long-term persistence in the upper airways, the virus is frequently co-detected with other respiratory pathogens. The diagnosis should therefore not be based only on qualitative PCR for HBoV1 DNA in respiratory samples [6, 20, 30]. Detection of HBoV1 DNA in blood, mRNA, and viral load assessment in airway samples and serology have been recommended as better tools for diagnostics to separate active HBoV1 infection from asymptomatic virus shedding [6, 7, 20, 21].

In this report, we present a case of a 17-month-old boy with typical symptoms of acute bilateral bronchiolitis: initially rhinorrhea and cough followed by difficulty in breathing and typical findings in auscultation, including wheezing and crepitation. Fever occurred for 2 days before hospitalization and his WBC was high with absolute granulocytosis. Because the latter findings pointed to complications, chest radiography was performed and right-sided pneumonia confirmed. The onset of the clinical course and normal range CRP value, were compatible with a viral LRTI.

NPA tested by multiplex PCR was positive only for HBoV1 DNA, while 18 other respiratory viruses, including those which often cause severe bronchiolitis in this age group (RSV, rhinovirus, and human metapneumovirus), were negative. It was not surprising that HBoV1 DNA was simultaneously found by PCR in all samples including stool. Viral passage through the gastrointestinal tract due to swallowing of respiratory secretions in patients with ARTIs has been suggested. By qPCR, a high viral load was detected in NPA and stool. Detection of HBoV1 exclusively, high HBoV1 DNA load in respiratory samples, and viremia are associated with a clinical picture of acute LRTI [6, 7]. However, in some other cases the viral DNA has remained detectable in blood for a prolonged time [15, 16]. In our patient, HBoV1 PCR was also positive in cell-free blood plasma, indicating that the virus was freed from the cells and that he had HBoV1 viremia. Diagnosis of acute HBoV1 infection is shown also by the presence of circulating HBoV1-specific IgM. Moreover, to verify that the current illness was in fact caused by acute HBoV1 infection, we looked for and found HBoV1 mRNA in PBMCs, verifying RNA transcription. To the best of our knowledge this is the first time HBoV1 mRNA has been detected in PBMCs, suggesting active replication in PBMCs. Existing data support that the detection of HBoV1 mRNA, at least in NPA, is more accurate than that of HBoV1 DNA, in the diagnosis of active infection [21, 31].

Out of the 424 samples analysed, 364 (85.8%) were positive for one or more viruses. Results are summarized in Table 2.The most commonly detected viruses were RSV, which was found in 129 (30.4%) patients and rhinoviruses in 116 (27.4%) accounting together for almost 60% of all detections. With moderate frequency have been detected HAdV in 31(7.3%) patients, influenza A in 28 (6.6%), HBoV in 24 (5.7%), enteroviruses and PIV 3 in 23 (5.4%) of patients respectively, and Influenza B in 21 (5.0%). A low frequency was exhibited by HMPV with 16 (3.8%) positive samples, human coronavirus OC43 with 13 (3.1%), PIV 1 with 12 (2.8%), PIV 4 with 9 (2.1%), PIV 2 with 7 (1.7%) and HCoV NL63 with 6 (1.4%). Coronavirus 229E could be detected only in a single sample.

Sample size was based on 95% confidence and 80% power to detect difference between rates of bacterial colonisation in symptomatic and asymptomatic individuals, if we assume 20% of HCWs will have bacterial colonization. These estimates are based on previous studies that describe adult colonisation rates. The sample size was calculated in Epi Info 2000. In order to allow for loss to follow up, 220 hospital staff members were to be recruited.

In Italy, the prevalence of exclusive breastfeeding at 3 months has been estimated to be nearly 60% (21). Considering this estimate, we calculated that a sample of 490 patients was sufficient to show an odds ratio of 0.6 for exclusively breastfed infants vs. infants with partial breastfeeding or artificial feeding, with a power of 80 and a 95% confidence level.

Proportions were compared using the Chi-square test or the Fisher exact test. Differences between means were studied through the Student's T-test. A P-value < 0.05 was considered statistically significant.

As the aim of our study was to analyse the effect of exclusive breastfeeding on the risk of respiratory infections, we decided to include in the same group artificial feeding + partial breastfeeding at symptom onset or at enrolment. A multivariable logistic regression analysis was performed in order to study the effect of exclusive breastfeeding (exclusive vs. partial breastfeeding or artificial feeding, at symptom onset for cases, or at enrolment for controls) and its duration (days) on the occurrence of respiratory tract infections, adjusted for the following variables: age (days), sex (male vs. female), ethnicity (caucasian vs. non caucasian), gestational age at birth (weeks), birth weight (kg), kind of delivery (vaginal vs. cesarean), parents' employment, parents' level of education (university degree vs. lower), parents' smoking habits, number of households, having at least 1 sibling.

Multicollinearity between the independent variables was assessed by studying the correlation matrix and examining the tolerance and the variance inflation factor (VIF).

Stata 13 was used for statistical analysis.

A possible correlation of virus prevalence and age of infection was assessed using univariate analyses. The Fisher’s exact test was used where cell counts below 5 were encountered; otherwise, the chi-squared test was performed. The same statistical tests were used to compare the frequency of subjects with single or multiple infections between age groups. In addition, Pearson correlation was used to examine co-infections of different viruses. All statistical analyses were performed using StataSE 12 (StatCorp. 2007. College Station, TX, USA).

Previous studies showed virus identification rate ranged from 32% to 85% of asthma exacerbation in children [14, 19]. The conservative estimation of virus detection rate of 50% would give the largest sample size estimate of 96 exacerbations with level of confidence at 95% and precision of detection rate of 10%. As the number of urgent visits due to asthma was 1.2 per person-year in children on regular inhaled steroids in another study, the number of subjects required would be around 80. We performed simple descriptive analyses of demographic data. The frequencies of presenting symptoms and physician diagnoses of unscheduled visits, virus detection rate and the distribution of different types of viruses were described. Student T test (+− Mann–Whitney U test) was used to compare continuous variables; for example, age and Pearson’s chi-square test (with Yates’correction/Fisher’s exact test) was used to compare categorical variables; for example, sex (female or male), atopic status (yes or no) between children with and without unscheduled visits. A p value less than 0.05 was considered to be statistically significant. All statistical analyses were carried out by the SPSS 11.0 software (SPSS Inc., Chicago, IL).

For detection of WUPyV-infections several conventional PCR formats as well as real-time PCR protocols were developed reporting sensitivities up to 100% and specificities up to 97.7% [10,23–25]. Antigen or antibody detection formats have not been reported, so far. No culture system is known at this time.

The diagnostics of established respiratory viruses is based on a broad spectrum of available assays, aiming at the detection of virus-specific antibodies, viral protein, or viral nucleic acids. Acute respiratory tract infections commonly have a short incubation period. Thus, antibodies are usually not present at the beginning of the acute phase of the disease. For this reason, detection of virus-specific antibodies by complement fixation assays, enzyme linked immunosorbent assays (ELISA), or immunofluorescence assays (IFA), all of which are available for established respiratory viruses, is inappropriate for the identification of the etiologic agent causing an acute respiratory tract infection, but useful for retrospective or epidemiological studies. Direct detection of viral protein or viral nucleic acids is the mode of choice for respiratory virus diagnostics. Viral cell culture represents the gold standard for the well established respiratory viruses, but is too slow for in time diagnostics. Less time consuming methods have been developed. These comprise polymerase chain reaction (PCR), antigen-specific ELISA, antigen-specific IFA, and, available only for a few respiratory viruses, very fast immunochromatographic assays. Sensitivity and specificity of these assays vary considerably. The PCR displays the highest sensitivity. Thus, being restricted to PCR formats for detection of WUPyV is no bias to sensitivity. Particularly real time PCR formats have an established track record in both, very sensitive detection and differentiation between colonisation and acute infection based on the quantitative data. Availability of automated extraction of nucleic acids permits short processing times and, by this, handling of large specimen cohorts.

For each enrolled patient (cases and controls), the following data were recorded: socio demographic data, gestational age, kind of delivery, birth weight, parents' level of education and employment, kind of feeding at symptom onset (exclusive breastfeeding, partial breastfeeding, artificial feeding), number of households, number of smokers in the family.

Data were collected through a questionnaire administered to parents of patients at enrolment, after signing an informed consent.

Epidemiological data were recorded in an electronic database (Microsoft Access).

Continuous data were expressed as median [first through third quartiles] and were compared using the Kruskall-Wallis test followed by pairwise Mann-Whitney test. Categorical data were expressed as number (percentages) and were evaluated using the chi-square test or Fisher’s exact test. p values less than 0.05 were considered significant. A univariate logistic regression with clinically relevant variables was used to identify variables associated with a complicated course. A multivariate conditional logistic regression, including variables with p value less than 0.10 in the previous step, was used to identify variables independently associated with complicated course. Similar statistical analyses were performed to identify variables independently associated with hospital death and mechanical ventilation for more than 7 days in survivors at day 28. Quantitative variables that did not validate the log-linearity assumption were transformed into categorical variable according to their median value. Missing data were imputed to the median or to the more frequent value. The accuracy of the final model was tested using area under the receiver operating characteristic curve analysis and the Hosmer-Lemeshow chi-square test. An additional multivariate conditional logistic regression, limited to bacterial and mixed groups, was performed to search specifically for an association between virus-bacteria coinfection and complicated course. Comparisons in the subgroup analysis of bacteria-matched patients involved univariate conditional logistic regression followed by multivariate conditional logistic regression to assess associations between microbiological diagnosis and complicated course, adjusting for clinically relevant variables. Analyses were performed using the SAS software package (SAS Institute, Cary, NC, USA).

During the successive 3.5-year period, 1229 patients with RTI were included in this study. The median age of the children was 8 months, varying from 1 month to 203 months, of which 63.1% of the patients were under 12 months (41.9% patients ≤6 months), 15.6% of the patients were between 1 and 2 years, 15.2% of the patients were between 2 and 5 years, whereas 6.0% of the patients were older than 5 years of age. Among the patients, there were 834 boys and 395 girls, and the sex ratio was 2.1:1. A potential viral pathogen was identified in 652 (53.1%) children (Table 1). For 1229 patients, PCR screening was performed for all 15 viruses and viral culture was performed for 7 viruses. The PCR results were positive for ≥1 virus in 652 (53.1%) children. The viral culture results were positive for ≥1 virus in 288 (23.4%) children. Notably, 266 (21.6%) of the patients had positive test results for ≥ 2 viruses.

Two new polyomaviruses were identified in 2007 in respiratory tract samples following large scale molecular screening using high throughput DNA sequencing of random clones and have been named after the institutes where they were found: KI (Karolinska Institute) polyomavirus (KIPyV) and WU (Washington University) polyomavirus (WUPyV). Data on seroprevalence indicate that infection is widespread ranging from 54.1 and 67% for KI and from 66.4% and 89% for WU in North American and German blood donors. Since their first identification, KI and WU viral sequences have been confirmed worldwide in respiratory samples from children with respiratory tract disease ranging from 0.2% to 2.7% and from 1.1 to 7%, respectively. However WUPyV and KIPyV were found at similar frequencies in control groups without respiratory diseases so the link between these polyomaviruses and acute respiratory diseases remains speculative.

Careful analysis is complicated by high co-infection rates with other well-characterized viral respiratory pathogens. A co-detection rate of 74% has been observed for KIPyV and rates ranging from 68 to 79% for WUPyV. Therefore, in a recent study in Southern China, hospitalized children with WUPyV infection displayed predominantly cough, moderate fever, and wheezing, but were also diagnosed with pneumonia, bronchiolitis, upper respiratory tract infections and bronchitis. As in most of infected children a single WUPyV infection was detected, it was suggested that the newly described polyomavirus can cause acute respiratory tract infection with atypical symptoms, including severe complications. Nevertheless these data have to be confirmed in further studies.

The presence of WUPyV and KIPyV in samples from children but not from immunocompetent adults suffering from LRTIs suggests that these viruses primarily infect the young population. A correlation between immunosuppression and reactivation of the two novel polyomaviruses has been suggested in immunocompromised patients and in AIDS patients at the molecular level, but no evidence of a role of these viruses as opportunistic pathogens has been given.

Overall, these data support the hypothesis that, in analogy with BK and JC polyomaviruses, KIPyV and WUPyV can establish persistent infection, and that virus replication may increase in immunocompromised hosts. However, in a recent study on immunocompetent and immunocompromised adult patients, real-time PCR detected KIPyV and WUPyV in 2.6% and 4.6% of HIV-1–infected patients respectively and in 3.1% and 0.8% of blood donors respectively, while no association was found between CD4+ cell counts in HIV-1 positive patients and infection with KIPyV or WUPyV.

KIPyV and WUPyV are also incidentally detected in adults with community acquired pneumonia, in immunocompromised hosts, and in patients with lung cancer; they are more often found in patients suffering an underlying medical condition and coinfections with KIPyV and WUPyV with other respiratory viruses are common. A recent study evaluating the prevalence and viral load of WUPyV and KIPyV in respiratory samples from immunocompromised and immunocompetent children showed that the prevalence of WUPyV and KIPyV is similar in hematology/oncology patients compared with that of the general pediatric population. High co-detection rates with other respiratory viruses, mainly RSV and enterovirus or rhinovirus, were found for WUPyV and KIPyV in both groups, in analogy with previous reports. However, higher viral loads for KIPyV in the immunocompromised group were detected, suggesting that there may be an increased replication of this virus in this population.

A similar association was not observed for WUPyV. Furthermore, in the immunocompromised group, infection with either virus occurred in older children compared with controls, which may indicate viral-reactivation Table 1.

Overall, in 70.6% of samples, one or more viruses were confirmed with the combined results of RT–PCR and DFA. The prevalence of RSV, FluA, FluB, PIV1, PIV3, and AdV according to DFA was 12%, 0.1%, 0.1%, 1.2%, 0.6%, and 1%, respectively. PIV2 was not detected during the study. Human coronavirus RNA was detected in 40 (6%; 95% CI: 4.3%–8.1%) of the 664 samples, 33/40 (82.5%) in nasopharyngeal swab, 6/40 (15%) in throat swabs and 1/40 (2.5%) in bronchoalveolar lavage. Of the 40 specimens positive for coronavirus, 21/40 (52.5%) were HKU1, 7/40 (17.5%) were OC43, 6/40 (15%) were 229E, and 6/40 (15%) were NL63. All specimens were also tested with real-time RT–PCR for hRV, HBoV, and hMPV. The most common virus detected was hRV (37.6%), followed by HBoV (20.9%) and hMPV (11.3%).

Most HCoV infections were detected in winter (22/40, 55%) and spring (10/40, 25%), whereas 6/40 (15%) positive samples were collected in autumn and only 2/40 (5%) in summer. The association between HCoV positivity and seasonality was statistically significant (P < 0.001). February had the highest number of HCoV-positive specimens (12/40, 30%), whereas HCoV was not detected in June or July (Figure 1).

Of the 40 HCoV-positive samples, only 12 (30%) were shown to involve only one virus, including 3/6 229E, 2/7 OC43, 5/21 HKU1, and 1/6 NL63. Species 229E was most frequently detected as a monoinfection (50%), but one or more other viruses were identified in the majority (28/40, 70%) of coronavirus-positive samples: 21/40 (52.5%) were dual infections, 6/40 (15%) were triple infections, and four viruses were detected simultaneously in one infection (2.5%). The viruses most frequently detected with HCoV were hRV (42.8%), HBoV (32.1%), RSV (28.6%), hMPV (21.4%), and AdV (3.6%).

On average, children with HCoV infections were older than those negative for HCoV (median ages in months: 23 vs 18 months, respectively), but infection with HCoV and age were not significantly associated (regardless of whether age was treated as a continuous or a categorical variable; data not shown). However, age was associated with the estimated quantity of HCoV viral nucleic acids present (measured as Ct). The association was nonlinear (P < 0.001) and the highest estimated viral load (lowest Ct) was detected in children around 10 months old. The estimated viral load increased with age for children younger than 10 months, whereas it decreased between 10 and 24 months. The estimated shape of the association is shown in Figure 2.

The statistical analyses were carried out using the SPSS 17.0 software package. The categorical variables were compared using the Chi-square test, and the continuous variables were compared using Student’s t-test or the nonparametric Mann–Whitney U-test. P-values <0.05 were considered to be significant.