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.
Beck
Eric T
ET
Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Jurgens
Lisa A
LA
Kehl
Sue C
SC
Bose
Michael E
ME
Patitucci
Teresa
T
LaGue
Elizabeth
E
Darga
Patrick
P
Wilkinson
Kimberly
K
Witt
Lorraine M
LM
Fan
Jiang
J
He
Jie
J
Kumar
Swati
S
Henrickson
Kelly J
KJ
D000818:Animals; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000302:isolation & purification; D009980:Influenza A virus / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology; D029524:Betainfluenzavirus / Q000302:isolation & purification*; D057230:Limit of Detection; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis*; D012136:Respiratory Syncytial Viruses / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D013552:Swine / Q000821:virology; D013997:Time Factors; D014481:United States / Q000453:epidemiology