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Journal of Clinical Microbiology, September 2009, p. 2772-2778, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00998-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rapid Multiplex Reverse Transcription-PCR Typing of Influenza A and B Virus, and Subtyping of Influenza A Virus into H1, 2, 3, 5, 7, 9, N1 (Human), N1 (Animal), N2, and N7, Including Typing of Novel Swine Origin Influenza A (H1N1) Virus, during the 2009 Outbreak in Milwaukee, Wisconsin{triangledown}

Jie He,1,2 Michael E. Bose,1,2 Eric T. Beck,1,2 Jiang Fan,1,2 Sagarika Tiwari,1,2 Jacob Metallo,1,2 Lisa A. Jurgens,1,2 Sue C. Kehl,1,3,4,5 Nathan Ledeboer,3,6 Swati Kumar,1,2,4,5 William Weisburg,7 and Kelly J. Henrickson1,2,4,5*

Midwest Respiratory Virus Program,1 Departments of Pediatrics,2 Pathology, Medical College of Wisconsin,3 Children's Research Institute,4 Children's Hospital of Wisconsin,5 Dynacare Laboratories, Milwaukee, Wisconsin,6 Nanogen, Inc., San Diego, California7

Received 19 May 2009/ Returned for modification 7 July 2009/ Accepted 23 July 2009

A large outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) infection in Milwaukee, WI, occurred in late April 2009. We had recently developed a rapid multiplex reverse transcription-PCR enzyme hybridization assay (FluPlex) to determine the type (A or B) and subtype (H1, H2, H3, H5, H7, H9, N1 [human], N1 [animal], N2, or N7) of influenza viruses, and this assay was used to confirm the diagnoses for the first infected patients in the state. The analytical sensitivity was excellent at 1.5 to 116 copies/reaction, or 10–3 to 10–1 50% tissue culture infective doses/ml. The testing of all existing hemagglutinin and neuraminidase subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low-level H3 cross-reaction with an H10N7 avian strain and only at 5.2 x 106 copies/reaction, not at lower concentrations. Comparisons of the FluPlex results with results from multiple validated in-house molecular assays, CDC-validated FDA-approved assays, and gene sequencing demonstrated 100% positive agreement for the typing of 179 influenza A viruses and 3 influenza B viruses, the subtyping of 110 H1N1 (S-OIV; N1 [animal]), 62 H1N1 (human), and 6 H3N2 (human) viruses, and the identification of 24 negative clinical samples and 100% negative agreement for all viruses tested except H1N1 (human) (97.7%). The small number of false-positive H1N1 (human) samples most likely represent increased sensitivity over that of other in-house assays, with four of four results confirmed by the CDC's influenza virus subtyping assay. The FluPlex is a rapid, inexpensive, sensitive, and specific method for the typing and subtyping of influenza viruses and demonstrated outstanding utility during the first 2 weeks of an S-OIV infection outbreak. Methods for rapid detection and broad subtyping of influenza viruses, including animal subtypes, are needed to address public concern over the emergence of pandemic strains. Attempts to automate this assay are ongoing.

* Corresponding author. Mailing address: Pediatrics/Infectious Disease/CCC/Suite c450, Children's Corporate Center, P.O. Box 1997, Milwaukee, WI 53201-1997. Phone: (414) 337-7073. Fax: (414) 337-7093. E-mail: Khenrick{at}mcw.edu

{triangledown} Published ahead of print on 29 July 2009.

Journal of Clinical Microbiology, September 2009, p. 2772-2778, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00998-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bose, M. E., Beck, E. T., Ledeboer, N., Kehl, S. C., Jurgens, L. A., Patitucci, T., Witt, L., LaGue, E., Darga, P., He, J., Fan, J., Kumar, S., Henrickson, K. J. (2009). Rapid Semiautomated Subtyping of Influenza Virus Species during the 2009 Swine Origin Influenza A H1N1 Virus Epidemic in Milwaukee, Wisconsin. J. Clin. Microbiol. 47: 2779-2786 [Abstract] [Full Text]  


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