Giuseppe
Emeritus
J Clin Microbiol. 2009 Jul 29. [Epub ahead of print]
Rapid Multiplex RT-PCR typing of influenza A and B, and subtyping of influenza A 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 current 2009 outbreak in Milwaukee, Wisconsin.
He J, Bose ME, Beck ET, Fan J, Tiwari S, Metallo J, Jurgens LA, Kehl SC, Ledeboer N, Kumar S, Weisburg W, Henrickson KJ. Midwest - Respiratory Virus Program (MRVP), Department of Pediatrics and Pathology, Medical College of Wisconsin, Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, Wisconsin, USA; Dynacare Laboratories, Milwaukee, Wisconsin, USA; Nanogen, Inc. San Diego, CA, USA.
A large outbreak of novel influenza (Flu) A (H1N1) virus [swine-origin influenza virus (S-OIV)] occurred in Milwaukee, Wisconsin in late April, 2009. We had recently developed a rapid multiplex RT-PCR enzyme hybridization assay (FluPlex) to type (A or B) and subtype [H1, H2, H3, H5, H7, H9, N1(human), N1(animal), N2, N7] influenza viruses that was used to confirm the first infected patients in the state. The analytical sensitivity was excellent at 1.5 - 116 copies/rxn or 10(-3) to 10(-1) TCID50/mL. Testing with all existing HA and NA subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low H3 cross reaction with an H10N7 avian strain and only at 5.2 x 10(6) copies/rxn not at lower concentrations. The FluPlex had positive agreements of 100% in typing 179 influenza A and subtyping 110 H1N1 (S-OIV, N1 animal), 62 H1N1 (human), 6 H3N2 (human), 3 Flu B, and 24 negative clinical samples, and negative agreement of 100% on all but H1N1 (human) (97.7%) compared to multiple validated "in-house" molecular assays, CDC validated FDA approved assays, and gene sequencing. The small number of "false positive" H1N1 (human) samples most likely represent increased sensitivity over other "in-house" assays with 4/4 confirmed by the CDC's Flu subtyping assay. The FluPlex is a rapid, inexpensive, sensitive and specific method for typing and subtyping influenza viruses and demonstrated outstanding utility during the first two weeks of an S-OIV outbreak. Methods for rapid diagnosis and broad subtyping of Flu viruses including animal subtypes are needed to address public concern over the emergence of pandemic strains. Attempts to automate this assay are ongoing.
PMID: 19641063 [PubMed - as supplied by publisher]
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Rapid Multiplex RT-PCR typing of influenza A and B, and subtyping of influenza A 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 current 2009 outbreak in Milwaukee, Wisconsin.
He J, Bose ME, Beck ET, Fan J, Tiwari S, Metallo J, Jurgens LA, Kehl SC, Ledeboer N, Kumar S, Weisburg W, Henrickson KJ. Midwest - Respiratory Virus Program (MRVP), Department of Pediatrics and Pathology, Medical College of Wisconsin, Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, Wisconsin, USA; Dynacare Laboratories, Milwaukee, Wisconsin, USA; Nanogen, Inc. San Diego, CA, USA.
A large outbreak of novel influenza (Flu) A (H1N1) virus [swine-origin influenza virus (S-OIV)] occurred in Milwaukee, Wisconsin in late April, 2009. We had recently developed a rapid multiplex RT-PCR enzyme hybridization assay (FluPlex) to type (A or B) and subtype [H1, H2, H3, H5, H7, H9, N1(human), N1(animal), N2, N7] influenza viruses that was used to confirm the first infected patients in the state. The analytical sensitivity was excellent at 1.5 - 116 copies/rxn or 10(-3) to 10(-1) TCID50/mL. Testing with all existing HA and NA subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low H3 cross reaction with an H10N7 avian strain and only at 5.2 x 10(6) copies/rxn not at lower concentrations. The FluPlex had positive agreements of 100% in typing 179 influenza A and subtyping 110 H1N1 (S-OIV, N1 animal), 62 H1N1 (human), 6 H3N2 (human), 3 Flu B, and 24 negative clinical samples, and negative agreement of 100% on all but H1N1 (human) (97.7%) compared to multiple validated "in-house" molecular assays, CDC validated FDA approved assays, and gene sequencing. The small number of "false positive" H1N1 (human) samples most likely represent increased sensitivity over other "in-house" assays with 4/4 confirmed by the CDC's Flu subtyping assay. The FluPlex is a rapid, inexpensive, sensitive and specific method for typing and subtyping influenza viruses and demonstrated outstanding utility during the first two weeks of an S-OIV outbreak. Methods for rapid diagnosis and broad subtyping of Flu viruses including animal subtypes are needed to address public concern over the emergence of pandemic strains. Attempts to automate this assay are ongoing.
PMID: 19641063 [PubMed - as supplied by publisher]
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