Likelihood that an unsubtypeable Influenza A result. in the Luminex xtag Respiratory Virus Panel. is indicative of novel A/H1N1 (swine-like) influenza
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1 JCM Accepts, published online ahead of print on 3 June 2009 J. Clin. Microbiol. doi: /jcm Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Likelihood that an unsubtypeable Influenza A result in the Luminex xtag Respiratory Virus Panel is indicative of novel A/H1N1 (swine-like) influenza Christine C. Ginocchio 1* and Kirsten St. George 2 1. North Shore-LIJ Health System Laboratories, 10 Nevada Drive, Lake Success, NY Wadsworth Center, New York State Department of Health, 5668 State Farm Road Slingerlands, Albany, NY Running title: Predictability of luminex xtag for novel H1N1 influenza *Corresponding author. Mailing address: Department of Laboratory Medicine, Division of Microbiology, Virology and Molecular Diagnostics, North Shore-LIJ Health System Laboratories, 10 Nevada Drive, Lake Success, NY Phone : , Fax: , cginocch@nshs.edu 1
2 With the sudden emergence of novel A/H1N1 (swine-like) influenza against a background of concurrently circulating seasonal H1N1 and H3N2 influenza viruses, diagnostic laboratories have been placed in a difficult situation regarding sensitive and accurate detection. This situation has been further complicated by the variable resistance patterns across the three circulating subtypes, making specific diagnosis important for treatment and prophylaxis, as well as for epidemiological tracking purposes. Many rapid EIA methods have been found to be unreliable for the detection of the new strain, and the delay in results with culture makes these methods suboptimal in many clinical and public health situations. Molecular amplification assays were, and continue to be, the method of choice for detection and subtyping of influenza viruses, in the current situation. Many laboratories lack the capability of developing molecular assays in-house, and currently the Luminex xtag Respiratory Virus Panel (RVP) assay (Luminex Molecular Diagnostics, Toronto, Canada) is the only FDA-approved assay that distinguishes the seasonal influenza A subtypes. The RVP assay detects influenza A by amplification of a target region on the matrix gene, and it distinguishes seasonal H1 and H3 subtypes using primers and probes specific for those hemagglutinin genes (2,3). In addition, the assay detects adenovirus, metapneumovirus, influenza B, parainfluenza viruses 1, 2, and 3, rhinovirus, and respiratory syncytial virus in a single reaction. The high degree of multiplexing afforded by the assay enables efficient use of sample, and minimizes labor relative to result output (1). When samples test positive in the RVP assay for influenza A, but negative for both H1 and H3 targets, they are classified as positive for novel influenza A. In studies performed at the North 2
3 Shore-LIJ Health System Laboratories (NS-LIJHS), the RVP subtyping results for 16 H1N1 strains and 86 H3N2 strains were compared to results obtained with conventional RT-PCR and sequencing; for the latter, the H1 and H3 specific primers had been designed at the Centers for Disease Control and Prevention (CDC). The RVP results correlated 100% with both the RT- PCR and sequencing results (unpublished data). For the current outbreak situation, we sought to assess the RVP method s reliability in accurately detecting and distinguishing the novel H1N1 (swine-like) influenza as an unsubtypeable influenza A virus, within the existing mixed background of seasonal H1N1 and H3N2 influenza. Over a period of 8 days starting on April 24, 2009, a total of 1,382 respiratory swab samples were received at the North Shore-LIJ Health System Laboratories (NS-LIJHS), Lake Success, NY, from primary care clinics, physician offices, and hospitals. Samples were initially tested with a variety of methods, including rapid antigen testing with either BinaxNOW A+B Test (Inverness Medical, Waltham, MA) or 3M Influenza A+B Rapid Test (3M Medical Diagnostics, St. Paul, MN) (n=1095); direct immunofluorescence with D3 Panel (Diagnostic Hybrids, Athens, OH) (n=1164); and R-Mix (Diagnostic Hybrids) rapid virus culture (n=1140). Samples testing positive for influenza A with any of these methods, or deriving from patients with a high potential to have the novel H1N1 (swine-like) influenza, were further tested with the RVP assay (n=375). Patient ages ranged from 10 months to 97 yr (median, 18 yr). A total of 201 of these samples were identified as influenza A positive by the RVP assay, two samples contained influenza B, and other respiratory viruses were detected in 58 samples (adenovirus, metapneumovirus, parainfluenza 1, 2, and 3, respiratory syncytial virus, rhinovirus). Of the 201 samples that tested positive for influenza A virus, two were positive for H1 (patient ages 8 and 3
4 ), 58 were positive for H3 (median patient age 25, range 10 mo to 90 yr), and 141 were negative on both hemagglutinin targets (median patient age 13, range 1 to 58 yr). Frozen residual portions of 101 of the 141 unsubtypeable influenza A virus-positive samples, and five samples identified as influenza A/H3, were forwarded to the Laboratory of Viral Diseases at the Wadsworth Center, Albany, NY for further testing. There they were tested with the real-time RT-PCR assay developed at the CDC for novel influenza A/H1N1 (swine-like) influenza. Incorporated in the test are real-time RT-PCR assays for influenza A, swine influenza A, and specific novel H1 hemagglutinin, as well as a control assay to assess specimen quality, extraction efficiency and presence of PCR inhibitors. The CDC protocol requires that each of the three influenza assays produce a positive real-time result, with CT values less than 37, if a sample is to be deemed positive for novel A/H1N1 (swine-like) influenza. A total of 99 of the 101 specimens that had tested as influenza A/non-subtypeable in the RVP assay, tested positive for novel H1N1 in the CDC assay, producing signals within range (CT < 37) on all three influenza target assays. For one of the remaining two specimens, weak positive signals (CT>37) were detected in two of the three influenza target assays, insufficient to classify the sample as positive for novel H1N1. The other specimen produced a weak positive result (CT> 37) in the influenza A assay only, and no detectable signal in the swine influenza A or novel H1 target assays. In both cases, there was insufficient sample left for further testing. Notably, both of these samples had produced relatively weak positive influenza A results in the RVP assay. The five samples that had tested positive for influenza A/H3 in the RVP assay were confirmed as positive for that influenza A subtype by a real-time RT-PCR assay, specific for the H3 hemagglutinin gene. 4
5 From these results, we consider the RVP assay to have demonstrated a high specificity for the detection of non-seasonal H1 or H3 influenza A viruses. During the current influenza outbreak, a test result of non-subtypeable influenza A obtained with this assay carries a high likelihood of being indicative of the presence of novel A/H1N1 (swine-like) influenza in the specimen. However, the patient should always be questioned regarding travel history and animal contact, with a view to determining potential exposures to other novel influenza agents. Acknowledgements The authors sincerely thank the staff of their laboratories for their long hours of work during the swine influenza outbreak that generated the data analyzed in this manuscript. CCG has received research funding and/or honoraria from Luminex, 3M and Diagnostic Hybrids. CCG is a member of the Scientific Advisory Board for Luminex. 5
6 107 References Krunic, N., T.D. Yager, D. Himsworth, F. Merante, S. Yaghoubian, and R Janeczko xtag RVP assay: analytical and clinical performance. J. Clin. Virol. 40 (Suppl. 1): S Mahony, J., S. Chong, F. Merante, S. Yaghoubian, T. Sinha, C. Lisle, and R. Janeczko Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay. J. Clin. Microbiol. 45: Merante, F., S. Yaghoubian, and R. Janeczko Principles of the xtag respiratory viral panel assay (RVP assay). J. Clin. Virol. 40 (Suppl. 1): S Downloaded from on April 7, 2019 by guest 6
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