Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis

To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT-PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. Respiratory syncytial viruses (A&B) were detectable in 45 of 84 (53.6%) nasopharyngeal aspirates from infants with bronchiolitis, whereas coronaviruses, influenza viruses, and parainfluenza viruses were not detectable in the same samples. Adenoviruses were detectable by PCR in 11 of 84 (13.1%) nasopharyngeal swabs. By using a picornavirus RT-PCR assay followed by a differential molecular hybridisation, rhinovirus and enterovirus RNA sequences were detected in 16 of 84 (19%) and in 10 of 84 (11.9%) of the nasopharyngeal swabs tested. Positive human rhinovirus or enterovirus RT-PCR assay, however, was the only evidence of respiratory infection in 8 of 84 (9.5%) and in 7 of 84 (8.33%) of the studied patients. Respiratory syncytial viruses, human rhinoviruses, adenoviruses, and enteroviruses occur in dual infections detected in 18 of 84 (21.4%) respiratory samples tested. The median duration of stay in hospital was not significantly different between the patients demonstrating a single viral infection and those with a dual viral infection (6.22 +/- 2.07 vs. 5. 04 +/- 0.95 days; P > 0.05). In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants.     

Prevalence of human metapneumovirus among hospitalized children younger than 1 year in Catalonia, Spain

Human metapneumovirus was discovered recently respiratory virus implicated in both upper and lower respiratory tract infection. In children, the clinical symptoms of human metapneumovirus are similar to those produced by respiratory syncytial virus, ranging from mild to severe diseases such as bronchiolitis and pneumonia. The aim of the present study was to describe the prevalence of human metapneumovirus and other common respiratory viruses among admitted to hospital infants. From January 2006 to June 2006, 99 nasopharyngeal aspirates were collected from hospitalized children younger than 12 months in order to study respiratory viruses. Human metapneumovirus detection was performed by cell culture and two RT-PCR targeting on polymerase and fusion genes. The latter gene was used for phylogenetic analysis. In 67/99 children (67%) at least one viral pathogen was identified, the viruses detected most frequently were respiratory syncytial virus (35%), human metapneumovirus (25%) and rhinovirus (19%). The results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among children less than 12 months; (2) children infected with human metapneumovirus were significantly older than those infected by respiratory syncytial virus; (3) human metapneumovirus was associated more frequently with pneumonia whereas respiratory syncytial virus was only detected in patients with bronchiolitis; (4) there was a clear epidemiological succession pattern with only a small overlap among the viruses detected most frequently; (5) all human metapneumovirus samples were clustered within sublineage A2.     

Human Bocavirus quantitative DNA detection in French children hospitalized for acute bronchiolitis

BACKGROUND: Human Bocavirus (HBoV) is a newly discovered parvovirus whose role as a causative agent of respiratory disease remains unclear. STUDY DESIGN: We investigated the presence of HBoV by quantitative PCR in the nasopharyngeal samples of 192 French children consecutively hospitalized for acute bronchiolitis. Other common respiratory viruses were detected using immunofluorescence assays, cell culture detection, or RT-PCR assays. RESULTS: HBoV was detected in 24 (12.5%) of 192 study children. In 14/192 cases (7%) HBoV was the sole isolate and in 10/192 (5%) it was part of a mixed viral infection. HBoV was the third most common pathogen detected after respiratory syncytial virus (45/192; 23%) and rhinovirus (24/192; 12%). It occurred more often in infants aged 1-12 months (P=0.002). Median levels of HBoV DNA genome in respiratory samples were significantly higher in patients with single HBoV infection than in patients with mixed respiratory viral infection with HBoV (4x10(8)copies/ml vs. 2x10(3)copies/ml, P<0.001). CONCLUSIONS: Our data suggest that HBoV at a high viral load could be an etiologic agent of respiratory tract disease, whereas the exact role of HBoV at a low viral load, as etiological cause or as pathophysiological co-factor of respiratory diseases, remains to be determined.     

Comparison of the FilmArray Respiratory Panel and Prodesse real-time PCR assays for detection of respiratory pathogens

We compared the diagnostic performance and overall respiratory pathogen detection rate of the premarket version of the FilmArray Respiratory Panel (RP) multiplex PCR assay (Idaho Technology, Inc., Salt Lake City, UT) with those of the Food and Drug Administration (FDA)-cleared Prodesse ProFlu+, ProFAST+, ProParaflu+, Pro hMPV+, and ProAdeno+ real-time PCR assays (Gen-Probe, San Diego, CA). The assays were performed on a panel of 192 nasopharyngeal-secretion specimens collected from 81 children under 1 year of age with upper respiratory tract symptoms. To resolve discordant results and confirm pathogens detected only by the larger FilmArray panel, we performed laboratory-developed real-time PCR assays. Among viruses detectable by both commercial assays (adenovirus, human metapneumovirus, influenza A virus, influenza B virus, parainfluenza viruses 1 to 3, and respiratory syncytial virus), the FilmArray and Prodesse assays showed good overall agreement (181/192 [94.3%]; kappa = 0.87; 95% CI, 0.79 to 0.94). FilmArray RP detected more parainfluenza viruses 1 and 3 than ProParaflu+ (18 versus 13) while ProAdeno+ detected more adenoviruses (11 versus 6), but these differences were not statistically significant. Additionally, FilmArray RP detected 138 pathogens (confirmed as true positives) not included in the Prodesse assays (rhinovirus [RV]/enterovirus [EV], 118; bocavirus, 8; coronavirus, 7; parainfluenza virus 4, 4; Mycoplasma pneumoniae, 1). FilmArray RP was cleared by the FDA following the completion of this study. The FDA-cleared version includes the following targets: adenovirus, coronaviruses HKU1 and NL63, human metapneumovirus (hMPV), influenza A virus (to type level only), influenza A H1 seasonal virus, influenza A H3 seasonal virus, influenza A virus H1-2009, influenza B virus, parainfluenza viruses 1 to 4, respiratory syncytial virus (RSV), and RV/EV (no differentiation). The larger panel in the FilmArray RP assay allowed the detection of additional respiratory pathogens compared to the Prodesse assays. In this population of young children with upper respiratory tract infection, RV/EV accounted for the majority of the additional pathogens detected by FilmArray RP.     

Molecular viral epidemiology and clinical characterization of acute febrile respiratory infections in hospitalized children in Taiwan

Acute respiratory infection (ARI) is a leading cause of morbidity and hospitalization in children. To profile the viruses causing ARI in children admitted to a community‐based hospital in central Taiwan, a cross‐sectional study was conducted on children under 14 years of age that were hospitalized with febrile ARI. Viral etiology was determined using conventional cell culture and a commercial respiratory virus panel fast assay (xTAG RVP), capable of detecting 19 different respiratory viruses and subtype targets. Demographic, clinical, and laboratory data were recorded and analyzed. The RVP fast assay identified at least one respiratory virus in 130 of the 216 specimens examined (60.2%) and rose to 137 (63.4%) by combining the results of cell culture and RVP fast assay. In order of frequency, the etiological agents identified were, rhinovirus/enterovirus (24.6%), respiratory syncytial virus (13.8%), adenovirus (11.5%), parainfluenza virus (9.2%), influenza B (8.4%), influenza A (5.4%), human metapneumovirus (4.6%), human coronavirus (2%), and human bocavirus (2%). Co‐infection did not result in an increase in clinical severity. The RVP assay detected more positive specimens, but failed to detect 6 viruses identified by culture. The viral detection rate for the RVP assay was affected by how many days after admission the samples were taken (P = 0.03). In conclusion, Rhinovirus/enterovirus, respiratory syncytial virus, and adenovirus were prevalent in this study by adopting RVP assay. The viral detection rate is influenced by sampling time, especially if the tests are performed during the first three days of hospitalization. J. Med. Virol. 87:1860–1866, 2015. © 2015 Wiley Periodicals, Inc.

     

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.     

Viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients

Syndromic diagnosis by multiplex nucleic acid amplification tests is the most practical approach to respiratory tract infections since the symptoms are rarely agent-specific. The aim of this study was to investigate the respiratory viruses in children admitted to a university hospital with acute respiratory tract infection during the last 8 years by a multiplex polymerase chain reaction (PCR) assay. A total of 3162 respiratory samples collected from children between April 2011 and April 2018 tested by a multiplex real-time PCR assay. Two different commercial assays were used during the study period, "AusDiagnostics/Respiratory Pathogens 12 (AusDiagnostics)" used between April 2011 and December 2015, which changed to "Fast Track Diagnostics/Respiratory Pathogens 21 (Fast Track Diagnostics)" after January 2016 to cover more viruses. Nucleic acid extraction was done by EZ1 Advanced XL platform (QIAGEN). Respiratory pathogens detected in 1857 of the 3162 (58.7%) samples. The most prevalent viruses during the 8-year period were rhinovirus/enterovirus (RV/EV; 36.2%), respiratory syncytial virus (RSV; 19%), and influenza virus A/B (14.7%). Rhinovirus was the main contributor to the RV/EV group as shown by the assay used during the 2016-2018 period. RV/EV and adenoviruses detected throughout the year. Influenza virus was most frequently detected during January to March when both RSV and metapneumovirus were also in circulation. The coinfection percentage was 10.2%. Rhinovirus was the most common virus in coinfections while RSV plus rhinovirus/enterovirus were the most frequent combination. RSV and metapneumovirus showed a similar seasonal distribution to the influenza virus, which made it necessary to use a virological diagnostic assay during the influenza season.     

Detection of viral, Chlamydia pneumoniae and Mycoplasma pneumoniae infections in exacerbations of asthma in children.

BACKGROUND: A high frequency of virus infections has been recently pointed out in the exacerbations of asthma in children. OBJECTIVES: To confirm this, using conventional and molecular detection methods, and expanding the study to younger children. STUDY DESIGN: One hundred and thirty-two nasal aspirates from 75 children hospitalized for a severe attack of asthma were studied (32 infants, mean age 9.1 months; and 43 children, mean age 5.6 years). According to the virus, a viral isolation technique, immunofluorescence assays (IFA) or both were used for the detection of rhinovirus, enterovirus, respiratory syncytial (RS) virus, adenovirus, coronavirus 229E, influenza and parainfluenza virus. Polymerase chain reaction (PCR) assays were used for the detection of rhinovirus, enterovirus, RS virus, adenovirus, coronavirus 229E and OC43, Chlamydia pneumoniae and Mycoplasma pneumoniae. RESULTS: Using IFA and viral isolation techniques, viruses were detected in 33.3% of cases, and by PCR techniques, nucleic acid sequences of virus, Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained in 71.9% of cases. The combination of conventional and molecular techniques detects 81.8% of positive samples. Two organisms were identified in the same nasal sample in 20.4% of the cases. The percentage of detections was higher (85.9%) in the younger group than in the other (77%). The most frequently detected agents were rhinovirus (46.9%) and RS virus (21.2%). Using PCR rather than conventional techniques, the detection rates were increased 5.8- and 1.6-fold in rhinovirus and RS virus infections, respectively. The detection levels of the other organisms are as follows: 9.8, 5.1, 4.5, 4.5, 4.5, 3.7, and 2.2% for enterovirus, influenza virus, Chlamydia pneumoniae, adenovirus, coronavirus, parainfluenza virus, and Mycoplasma pneumoniae, respectively. CONCLUSION: These results confirm the previously reported high frequency of rhinovirus detection in asthmatic exacerbations in children. They also point out the frequency of RS virus detection, and emphasize the fact that PCR assays may be necessary to diagnose respiratory infections in asthma.     

Epidemiological and phylogenic study of human metapneumovirus infections during three consecutive outbreaks in Normandy, France

Human metapneumovirus (hMPV) is responsible for respiratory tract disease, particularly in the young and elderly population. An epidemiological and phylogenic study was performed on children admitted to hospital with an acute lower respiratory tract infection (LRI). Data were obtained and analyzed over three consecutive winters, from 2002-2003 to 2004-2005. Each year during the winter period, from November to March, 2,415 nasal swabs were tested by a direct immunofluorescence assay (DFA) for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses, and adenoviruses. Rhinoviruses, enteroviruses, and coronaviruses OC43 and 229E were detected by RT-PCR. A RT-PCR designed for the M gene was performed on negative samples for hMPV detection and phylogenic analyses. For the three consecutive winters, hMPV represented 10%, 22.6%, and 8.8% of virus-negative samples, respectively. In most cases, clinical symptoms indicated a LRI with a final diagnosis of bronchiolitis. During the winter of 2003-2004, all viral clusters (A1, A2, B1, and B2) that circulated in France shifted progressively from the A group to the B group. This study determined the prevalence of hMPV in Normandy, its clinical impact and permitted the analysis of the molecular evolution during the successive outbreaks.     

Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens Basic Kit.

BACKGROUND: Molecular methods based on RNA amplification are needed for sensitive detection of enteroviruses in clinical samples. Many 'in house' methods based on reverse-transcribed PCR (RT-PCR) could be difficult to use in the routine diagnostic laboratory since they tend to be time-consuming, use reagents from many different suppliers and include non-routine procedures. OBJECTIVES: The aim of this study was to develop and evaluate methods based on nucleic acid sequence based amplification (NASBA) for detection of enterovirus sequences. STUDY DESIGN: 'In house' prepared and commercially available reagents were utilised to develop enterovirus-specific NASBA assays. Optimised methods were evaluated using clinical samples (cerebrospinal fluid, respiratory and stool samples), titred virus controls and in vitro produced synthetic RNA. Results for NASBA were compared with RT-PCR and virus culture. RESULTS: Kit-based reagents gave an equivalent sensitivity to the more laborious 'in house' molecular assays (NASBA and RT-PCR) on clinical material and controls. All molecular methods picked up enterovirus positive clinical samples that were not identified by culture. End point detection sensitivity for the NASBA assay based on the NucliSens Basic Kit was 相似文献   

Prevalence of viral respiratory tract infections in children with asthma

BACKGROUND: Previous studies support a strong association between viral respiratory tract infections and asthma exacerbations. The effect of newly discovered viruses on asthma control is less well defined. OBJECTIVE: We sought to determine the contribution of respiratory viruses to asthma exacerbations in children with a panel of PCR assays for common and newly discovered respiratory viruses. METHODS: Respiratory specimens from children aged 2 to 17 years with asthma exacerbations (case patients, n = 65) and with well-controlled asthma (control subjects, n = 77), frequency matched by age and season of enrollment, were tested for rhinoviruses, enteroviruses, respiratory syncytial virus, human metapneumovirus, coronaviruses 229E and OC43, parainfluenza viruses 1 to 3, influenza viruses, adenoviruses, and human bocavirus. RESULTS: Infection with respiratory viruses was associated with asthma exacerbations (63.1% in case patients vs 23.4% in control subjects; odds ratio, 5.6; 95% CI, 2.7- 11.6). Rhinovirus was by far the most prevalent virus (60% among case patients vs 18.2% among control subjects) and the only virus significantly associated with exacerbations (odds ratio, 6.8; 95% CI, 3.2-14.5). However, in children without clinically manifested viral respiratory tract illness, the prevalence of rhinovirus infection was similar in case patients (29.2%) versus control subjects (23.4%, P > .05). Other viruses detected included human metapneumovirus (4.6% in patients with acute asthma vs 2.6% in control subjects), enteroviruses (4.6% vs 0%), coronavirus 229E (0% vs 1.3%), and respiratory syncytial virus (1.5% vs 0%). CONCLUSION: Symptomatic rhinovirus infections are an important contributor to asthma exacerbations in children. CLINICAL IMPLICATIONS: These results support the need for therapies effective against rhinovirus as a means to decrease asthma exacerbations.     

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.     

The role of influenza,RSV and other common respiratory viruses in severe acute respiratory infections and influenza-like illness in a population with a high HIV sero-prevalence,South Africa 2012–2015

BackgroundViruses detected in patients with acute respiratory infections may be the cause of illness or asymptomatic shedding.ObjectiveTo estimate the attributable fraction (AF) and the detection rate attributable to illness for each of the different respiratory virusesStudy designWe compared the prevalence of 10 common respiratory viruses (influenza A and B viruses, parainfluenza virus 1–3; respiratory syncytial virus (RSV); adenovirus, rhinovirus, human metapneumovirus (hMPV) and enterovirus) in both HIV positive and negative patients hospitalized with severe acute respiratory illness (SARI), outpatients with influenza-like illness (ILI), and control subjects who did not report any febrile, respiratory or gastrointestinal illness during 2012–2015 in South Africa.ResultsWe enrolled 1959 SARI, 3784 ILI and 1793 controls with a HIV sero-prevalence of 26%, 30% and 43%, respectively. Influenza virus (AF: 86.3%; 95%CI: 77.7–91.6%), hMPV (AF: 85.6%; 95%CI: 72.0–92.6%), and RSV (AF: 83.7%; 95%CI: 77.5–88.2%) infections were associated with severe disease., while rhinovirus (AF: 46.9%; 95%CI: 37.6–56.5%) and adenovirus (AF: 36.4%; 95%CI: 20.6–49.0%) were only moderately associated.ConclusionsInfluenza, RSV and hMPV can be considered pathogens if detected in ILI and SARI while rhinovirus and adenovirus were commonly identified in controls suggesting that they may cause only a proportion of clinical disease observed in positive patients. Nonetheless, they may be important contributors to disease.     

Identification of enteroviral infection among infants and children admitted to hospital with acute gastroentritis in Ho Chi Minh City, Vietnam

A total of 276 fecal specimens collected from infants and children admitted to hospital with acute gastroenteritis in Ho Chi Minh City, Vietnam from October 2002 to September 2003, were tested for the presence of enteroviruses by RT-PCR and virus isolation. Enteroviruses were detected in 27 patients by RT-PCR corresponding to 9.8%. However, only four enterovirus strains could be isolated by cell culture with two different cell lines CaCo2 and Vero, showing specific cytopathic effect (CPE). The results clearly indicate that RT-PCR is a sensitive, specific assay to investigate the true burden of acute gastroenteritis due to enteroviruses in clinical fecal specimens. In the present study, enteroviruses were identified throughout the year except in May and the highest number was in December. Enteroviruses were subjected to molecular analysis by sequencing. It was found that enterovirus strains detected were classified further into two distinct genetic clusters (I, II) and demonstrated the great genetic diversity among them. Based on genetic analysis, 5' noncoding region (5' NCR) sequences suggested the predominant presence of Vietnamese enteroviruses with the greatest similarities to coxsakieviruses (51.9%) and echoviruses (29.6%). Interestingly, two of the sequenced specimens of enteroviruses were similar to a new strain called enterovirus 74. This report is the first detection of enteroviral infection in feces from infants and children admitted to hospital with acute gastroenteritis in Vietnam.     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

Performance of the Luminex xTAG Respiratory Viral Panel Fast in a clinical laboratory setting

The aim of the study was to develop a real-time RT-PCR for the detection of enteroviruses (EVs) and rhinoviruses (RVs) and to assess the performance of the xTAG RVP Fast assay in comparison to a direct fluorescent assay (DFA), a real-time RT-PCR assay for the detection of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), and the EV/RV RT-PCR assay developed in this study. The performance of the RVP Fast assay was assessed in the analysis of 373 nasopharyngeal samples. For the viruses of the DFA panel, detection rates of 27.6% and 23.8% were obtained by RVP and DFA, respectively, in analysis of a set of 297 samples collected in 2009-2010. These results show statistically significant superiority of the RVP Fast assay (P=0.049). For RSV, hMPV, EV, and RV, detection rates of 48.0% and 45.2% were achieved by RVP and RT-PCR, respectively. For individual targets, increased detection of EV/RV (P=0.043) and decreased detection of influenza A virus (P=0.004) by RVP in comparison to real-time RT-PCR was observed. The results of the present study imply the need to adjust the InfA component of the RVP Fast assay to also cover the InfA(H1N1) 2009 virus.