Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin.

Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10(-2) to 10(1) TCID(50)/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >10(7) TCID(50)/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other "in-house" validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.

Rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza A H1N1 virus epidemic in Milwaukee, Wisconsin.

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.