Switching Gears for an Influenza Pandemic: Validation of a Duplex Reverse Transcriptase PCR Assay for Simultaneous Detection and Confirmatory Identification of Pandemic (H1N1) 2009 Influenza Virus

Pigs and humans have many similarities with respect to influenza viruses. Both have well-established, distinct, and stable lineages of influenza A virus that cause periodic epidemics associated with morbidity and mortality (21). The swine influenza A viruses currently circulating in North America are triple reassortants that have components of avian, human, and swine origin (19). For pandemic (H1N1) 2009 influenza virus, the PB2 and PA polymerase components are derived from avian influenza virus lineages; PB1 is of human influenza virus origin; and the genes encoding hemagglutinin (HA), neuraminidase, nucleoprotein, matrix protein (M), and nonstructural protein are of swine lineages of influenza A virus (16). Sporadic cases of human infection with triple-reassortant swine influenza viruses were previously documented; however, until recently, human-to-human transmission was not sustained (6, 16, 19). In March 2009, Mexico reported clusters of a respiratory disease that was subsequently identified as pandemic (H1N1) 2009 influenza virus. As of 30 August 2009, over 250,000 cases and 2,837 deaths had been documented world- wide (23). This led the WHO to increase the pandemic alert to phase 6 and declare an international public health emergency.

Rapid diagnosis by molecular methods, such as reverse transcriptase PCR (RT-PCR) assays, is the cornerstone of planning for a pandemic. As outlined in the Canadian Pandemic Influenza Plan (18), provincial public health or designated laboratories should have the capacity to identify and subtype influenza viruses using molecular methods. The high sensitivity and specificity of RT-PCR compared to those of conventional detection methods prompted many laboratories to implement RT-PCR for the detection of influenza viruses. With increasing rates of antiviral resistance in circulating seasonal human influenza A virus strains (H1N1 and H3N2), RT-PCR is now being used for influenza A virus subtyping in order to facilitate the clinical management of patients (9). Influenza A viruses that cannot be subtyped must be forwarded to reference laboratories to rule out the presence of a novel influenza virus strain (3, 18). This was the scenario that played out in the Capital District Health Authority (CDHA) microbiology laboratory in Haliax, Nova Scotia, Canada, and many other laboratories across North America.