Rapid Semiautomated Subtyping of Influenza Virus Species during the 2009 Swine Origin Influenza A H1N1 Virus Epidemic in Milwaukee, Wisconsin

Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.
Journal of clinical microbiology (Impact Factor: 3.99). 07/2009; 47(9):2779-86. DOI: 10.1128/JCM.00999-09
Source: PubMed


In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Seasonal), employing the NucliSENS easyMAG system (bioMérieux, Durham, NC) and a Raider thermocycler (HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demonstrated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and 100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiautomated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also demonstrates the importance of quickly distributing information on new agents and of having rapid influenza virus subtyping assays widely available for clinical and public health decisions.

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    • "After the worldwide outbreak of pandemic influenza A/H1N1 2009 (A/H1N1pdm), many diagnostic methods for detecting the causative virus, including the rRT-PCR assay, were established by many groups (Beck et al., 2010; Bose et al., 2009; Carr et al., 2009; Chidlow et al., 2010; Dong et al., 2010; Ge et al., 2009; Gunson et al., 2010; Hall et al., 2009; He et al., 2009; Jiang et al., 2010; Kubo et al., 2010; Lau et al., 2009; LeBlanc et al., 2009; Liu et al., 2009; Pabbaraju et al., 2009; Poon et al., 2009; Wang et al., 2009; Wenzel et al., 2009; Whiley et al., 2009; Wu et al., 2010; Yang et al., 2009). Type A rRT-PCR and improved H1pdm rRT-PCR assays, as well as the newly developed H1 rRT-PCR and H3 rRT-PCR assays used for subtyping human seasonal influenza A viruses, were shown to have good linearity (R 2 = 0.99) and high sensitivity (Tables 2–4 and Fig. 1). "
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