Twelve years' detection of respiratory viruses by immunofluorescence in hospitalised children: impact of the introduction of a new respiratory picornavirus assay

The study was approved by the Ethics Committee of the University Hospital of Bern in accordance with cantonal ethical regulations (Nr. E 10-01-10). The study included consecutive respiratory tract samples from children under the age of 17 years, who were hospitalized at the Department of Pediatrics, University Hospital Bern, between May 1^st 1998 and April 30^th 2010. During the entire study period the pediatrics department had the policy of screening children for respiratory viruses if they were hospitalized with a respiratory illness or if they developed respiratory symptoms during their hospital stay.

A total of 12'189 respiratory samples were collected. After exclusion criteria, 10'629 samples remained for the retrospective analysis of DFA results. The exclusion criteria were as follows: samples other than nasopharyngeal aspirates, samples containing less than 20 epithelial cells and samples not tested against the whole viral panel; results of samples from the same patient taken within a time period of 7 days (considered part of the same respiratory episode); results for the month of August 2009, since during this time period practically all respiratory samples were tested by PCR rather than DFA due to the influenza A H1N1 pandemic.

All samples were analysed at the Institute for Infectious Diseases, University of Bern. The methods used have previously been described [24].

Between May 1^st 1998 and August 31^st 2007, the Light Diagnostics Respiratory Viral Screen DFA (Chemicon International, now Millipore) and single fluorescein-conjugated monoclonal antibodies against ADV, RSV, IFA, IFB and PIV 1-3 were used. From September 1^st 2007 to April 30^th 2010, the D3 Ultra DFA Respiratory Virus Screening & ID Kit (Diagnostic Hybrids) was used for the same viruses. Starting in March 2006, the Light Diagnostics Pan-Enterovirus Reagent "Blend" (Chemicon International/Millipore) was introduced for the detection of respiratory picornaviruses. This assay is formally an indirect immunofluorescence assay, as described elsewhere [24], and does not allow the differentiation between rhinoviruses and enteroviruses. In November 2006, the DFA Metapneumovirus Identification Kit (Diagnostic Hybrids) was added to the screening.

An epidemiological year was defined as May 1^st to April 30^th of the following year. Summer was defined as the months July to September, and winter as January to March. Epidemiological years were designated "odd" if the month of January was in an odd year, and they were labelled "even" if the month of January was in an even year.

All statistical analyses were performed with the GraphPad Prism 5^® software tool (GraphPad Software, Inc.). Proportions were compared using the chi-square test. Medians were compared with the Kruskal-Wallis test and Dunn's multiple comparison test. A cut-off of p ≤ 0.05, two tailed, was used for all statistical analyses.

A total of 10'629 samples from 8'285 patients were analysed. The median age of the study population was 11 months (range 0-17 years) and 57.5% were boys.

Before the addition of DFA for picornavirus and hMPV, the rate of viral detection was dominated by the RSV season, with a yearly average rate of 35%, peaks of up to 64% (average 46%) in winter seasons, and troughs as low as 4% (average 16%) during summer time. The addition of DFA for picornaviruses increased the positivity rate, and dampened the seasonal variations. The positivity rate after 2006 was on average 65% in winter and 52% in summer, with a yearly average of 58% (versus 35%; p < 0.0001) (Figure 1, Table 1). For comparison, analysis performed on 256 specimens in parallel to DFA screening with the xTag Respiratory Viral Panel (Luminex Molecular Diagnostics) between November 2006 and September 2007 yielded a positivity rate of 78%. The higher detection rate by PCR could mostly be attributed to increased detection of respiratory picornaviruses; 57 from the 78 additional positive results were respiratory picornaviruses (unpublished data).

Respiratory picornaviruses were the most common pathogens detected overall in our study population after the introduction of the assay (27% versus 21% for RSV; p < 0.0001) (Table 1). They were present year round, with peaks in the spring and the fall. During the summer time, respiratory picornaviruses also accounted for the majority of viral respiratory infections (Figure 1). Low prevalence of respiratory picornaviruses during winter time coincided with the winter peaks caused by RSV, influenza, or hMPV, and this was the only time during the year when respiratory picornaviruses were not the most commonly detected respiratory virus (Figure 2).

RSV was the second most commonly detected pathogen after picornaviruses (overall prevalence of 21%), but the most prevalent virus during the winter months (Table 1, Figure 2). It manifested a biennial pattern, with large winter seasons in odd years alternating with smaller ones in even years.

Influenza A virus was found in 4.1% of all samples and caused yearly winter epidemics, except during the winter season 2005-2006 when it was replaced by influenza B virus (Table 1, Figure 2).

hMPV was detected in 3.4% of all samples collected after November 2006 (Table 1). Yearly hMPV activity varied from being almost absent during the winter season 2006 to 2007, to causing yearly winter outbreaks during the following years (Figure 2, Figure 3). In early 2010, viral activity surpassed previous years, with more cases observed within 5 months (71 cases between December 2009 and April 2010) than during the entire previous period since introduction of the DFA test (56 cases between November 2006 and November 2009).

We compared the proportion of samples positive for a given virus by age (Figure 4). Respiratory picornaviruses were the most common pathogens in children ≥1 years (1-4 years: 33% versus 15% RSV, p < 0.0001; 5-8 years: 26% versus <7% other viruses, p < 0.0001; 9-17 years: 16% versus 9% influenza A, p = 0.007), and RSV was the most common detected in children < 1 year (30% versus 23% picornavirus, p < 0.0001). Influenza A showed growing importance with age, and was the second most common virus detected in children >9 years old (9% influenza A versus <4% other viruses, p < 0.0001).

Out of the total of 10'629 samples analysed, 82 were positive for two viruses (Table 2). No sample was positive for more than two viruses. This corresponds to a codetection rate of 0.8% with DFA.

With the xTag Respiratory Viral Panel (Luminex) 10.2% of samples were positive for two viruses (in 81% of these a respiratory picornavirus was present). In 0.8% of samples we detected three respiratory viruses.