title: Rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza A H1N1 virus epidemic in Milwaukee, Wisconsin.
abstract: 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.
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Bose
authors.forename: Eric T
authors.initials: ET
authors.lastname: Beck
authors.forename: Nate
authors.initials: N
authors.lastname: Ledeboer
authors.forename: Sue C
authors.initials: SC
authors.lastname: Kehl
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Jurgens
authors.forename: Teresa
authors.initials: T
authors.lastname: Patitucci
authors.forename: Lorraine
authors.initials: L
authors.lastname: Witt
authors.forename: Elizabeth
authors.initials: E
authors.lastname: LaGue
authors.forename: Patrick
authors.initials: P
authors.lastname: Darga
authors.forename: Jie
authors.initials: J
authors.lastname: He
authors.forename: Jiang
authors.initials: J
authors.lastname: Fan
authors.forename: Swati
authors.initials: S
authors.lastname: Kumar
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Henrickson
mesh_terms: D001331:Automation; D017931:DNA Primers / Q000235:genetics; D004196:Disease Outbreaks; D005838:Genotype; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000302:isolation & purification; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000453:epidemiology* / Q000821:virology*; D015203:Reproducibility of Results; D012136:Respiratory Syncytial Viruses / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D014922:Wisconsin / Q000453:epidemiology
chemical_list: D017931:DNA Primers