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Journal of Clinical Microbiology, July 2009, p. 2347-2348, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.01027-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Likelihood that an Unsubtypeable Influenza A Virus Result Obtained with the Luminex xTAG Respiratory Virus Panel Is Indicative of Infection with Novel A/H1N1 (Swine-Like) Influenza Virus{triangledown}

Christine C. Ginocchio* Kirsten St. George

North Shore-LIJ Health System Laboratories
10 Nevada Drive
Lake Success, NY 11042

Wadsworth Center
New York State Department of Health
Empire State Plaza
P.O. Box 509
Albany, NY 12201-0509

Received 24 May 2009/ Accepted 29 May 2009


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INTRODUCTION
 
With the sudden emergence of novel A/H1N1 (swine-like) influenza virus 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 enzyme immunoassay methods have been found to be unreliable for the detection of the new strain, and the delay in results obtainable with culture makes these methods suboptimal in many clinical and public health situations. Molecular amplification assays have been, 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 Food and Drug Administration-approved assay that distinguishes the seasonal influenza A virus subtypes. The RVP assay detects influenza A virus by amplification of a target region on the matrix gene, and it distinguishes seasonal H1 and H3 virus subtypes by the use of primers and probes specific for those hemagglutinin genes (2, 3). In addition, the assay detects adenovirus, metapneumovirus, influenza B virus, 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 the output (1).

When samples test positive in the RVP assay for influenza A virus but negative for both H1 and H3 targets, the results are classified as positive for novel influenza A virus. In studies performed at the North Shore-LIJ Health System Laboratories, the RVP subtyping results for 16 H1N1 strains and 86 H3N2 strains were compared to results obtained with conventional reverse transcriptase PCR (RT-PCR) and sequencing; for the latter, the H1- and H3-specific primers used 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 reliability of the RVP method for accurately detecting and distinguishing the novel H1N1 (swine-like) influenza virus as an unsubtypeable influenza A virus within the existing mixed background of seasonal H1N1 and H3N2 influenza virus strains.

Over a period of 8 days starting on 24 April 2009, a total of 1,382 respiratory swab samples from primary care clinics, physician offices, and hospitals were received at the North Shore-LIJ Health System Laboratories, Lake Success, NY. Samples were initially tested with a variety of methods, including rapid antigen testing with either the BinaxNOW A+B test (Inverness Medical, Waltham, MA) or the 3M Rapid Detection Flu A+B test (3M Medical Diagnostics, St. Paul, MN) (n = 1,095), direct immunofluorescence with a D3 panel (Diagnostic Hybrids, Athens, OH) (n = 1,164), and R-Mix (Diagnostic Hybrids) rapid virus culture (n = 1,140). Samples testing positive for influenza A virus with any of these methods, or deriving from patients with a high potential of harboring the novel H1N1 (swine-like) influenza virus, were further tested with the RVP assay (n = 375). Patient ages ranged from 10 months to 97 years of age (median, 18 years). A total of 201 of these samples were identified as influenza A virus positive by the RVP assay, two samples contained influenza B virus, and other respiratory viruses (adenovirus, metapneumovirus, parainfluenza types 1, 2, and 3, respiratory syncytial virus, and rhinovirus) were detected in 58 samples. Of the 201 samples that tested positive for influenza A virus, 2 were positive for H1 (patient ages 8 and 16 years), 58 were positive for H3 (median patient age, 25 years [range, 10 months to 90 years]), and 141 were negative on both hemagglutinin targets (median patient age, 13 years [range, 1 to 58 years]). Frozen residual portions of 101 of the 141 unsubtypeable influenza A virus-positive samples, and five samples identified as representing influenza A/H3 virus, 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 virus. Incorporated in the test are real-time RT-PCR assays for influenza A virus, swine influenza A virus, and specific novel H1 hemagglutinin, as well as a control assay to assess specimen quality, extraction efficiency, and the presence of PCR inhibitors. The CDC protocol requires that each of the three influenza assays must produce a positive real-time result, with threshold cycle (CT) values of less than 37, if a sample is to be deemed positive for novel A/H1N1 (swine-like) influenza virus.

A total of 99 of the 101 specimens that had tested as representing influenza A virus or as unsubtypeable in the RVP assay tested positive for the novel H1N1 virus in the CDC assay, producing signals within range (CT < 37) in 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 virus target assays, which were results that were insufficient to classify the sample as positive for novel H1N1 virus. The other specimen produced a weak positive result (CT > 37) in the influenza A virus assay only and no detectable signal in the swine influenza A virus or novel H1 virus 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 virus results in the RVP assay. The five samples that had tested positive for influenza A/H3 virus in the RVP assay were confirmed to be positive for that influenza A virus subtype by a real-time RT-PCR assay specific for the H3 hemagglutinin gene.

From these results, we consider the RVP assay to have demonstrated high specificity for the detection of nonseasonal H1 or H3 influenza A viruses. During the current influenza outbreak, a test result positive for unsubtypeable influenza A virus obtained with this assay carries a high likelihood of being indicative of the presence of novel A/H1N1 (swine-like) influenza virus 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.


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ACKNOWLEDGMENTS
 
We sincerely thank the staff of our laboratories for their long hours of work during the swine influenza outbreak that generated the data analyzed in this work.

C.C.G. has received research funding and/or honoraria from Luminex, 3M, and Diagnostic Hybrids. C.C.G. is a member of the Scientific Advisory Board for Luminex.


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FOOTNOTES
 
* Phone: (516) 719-1079, Fax: (516) 719-1254, E-mail: cginocch{at}nshs.edu Back

{triangledown} Published ahead of print on 3 June 2009. Back


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REFERENCES
 
    1
  1. Krunic, N., T. D. Yager, D. Himsworth, F. Merante, S. Yaghoubian, and R. Janeczko. 2007. xTAG RVP assay: analytical and clinical performance. J. Clin. Virol. 40(Suppl. 1):S39-S46.[CrossRef][Medline]
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  2. Mahony, J., S. Chong, F. Merante, S. Yaghoubian, T. Sinha, C. Lisle, and R. Janeczko. 2007. 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. 45:2965-2970.[Abstract/Free Full Text]
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  3. Merante, F., S. Yaghoubian, and R. Janeczko. 2007. Principles of the xTAG respiratory viral panel assay (RVP assay). J. Clin. Virol. 40(Suppl. 1):S31-S35.[CrossRef][Medline]

Journal of Clinical Microbiology, July 2009, p. 2347-2348, Vol. 47, No. 7
0095-1137/09/$08.00+0     doi:10.1128/JCM.01027-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Vinikoor, M., Stevens, J., Nawrocki, J., Singh, K. (2009). Influenza A Virus Subtyping: Paradigm Shift in Influenza Diagnosis. J. Clin. Microbiol. 47: 3055-3056 [Full Text]  

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