Switching gears for an influenza pandemic: validation of a duplex RT-PCR for 2 simultaneous detection and confirmation of pandemic (H1N1) 2009.


Rapid methods for detection and confirmation of pandemic influenza A [also known as pandemic (H1N1) 2009] are of utmost importance. In this study, a conventional reverse-transcriptase polymerase chain reaction (RT-PCR) was designed, optimized and validated for the detection of influenza A and the hemagglutinin of swine lineage H1 (swH1). Nucleic acids were extracted from 198 consecutive nasopharyngeal, nasal or throat swabs collected early in the outbreak [127 negatives, 66 pandemic (H1N1) 2009, 3 seasonal influenza A (H1N1) and 2 seasonal influenza A (H3N2)]. The performance characteristics of duplex RT-PCR were assessed compared to various detection methods: monoplex RT-PCR at the National Microbiology Laboratory (NML), a real-time RT-PCR using a Center for Disease Control and Prevention (CDC) protocol, a in-house multiplex RT-PCR [targeting influenza A, influenza B and respiratory syncytial virus (RSV)], and a rapid antigen test, the BinaxNOW Influenza A & B. For influenza A detection, the sensitivity of duplex RT-PCR was 97.2%, versus 74.6%, 71.8%, 47.8%, and 12.7% for the other assays, respectively. Duplex RT-PCR was also able to identify swH1 in the 94% of cases, thereby reducing the number of specimens forwarded to reference laboratories for confirmation. Only a limited number of influenza A-positive specimens fell below the limit of detection of the swH1 primers. Overall, duplex RT-PCR is a reliable method for simultaneous detection and confirmation of pandemic (H1N1) 2009 and would be particularly attractive to laboratories without real-time RT-PCR capability.


Pigs and humans have many similarities with respect to influenza virus. Both have well-established, distinct and stable lineages of influenza A virus that cause periodic epidemics associated with morbidity and mortality (21). Swine influenza A viruses currently circulating in North America are triple re-assortants that have components of avian, human and swine origin (19). For pandemic (H1N1) 2009, the polymerase components PB2 and PA are derived from avian influenza lineages, PB1 is of human influenza origin, and the genes encoding hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M), and nonstructural protein (NS) are of swine lineages of influenza A (16). Sporadic cases of humans infection with triple re-assortant swine influenza viruses had been previously documented; however, until recently human-to-human transmission was not sustained (6, 16, 19). In March 2009, Mexico reported clusters of respiratory disease that was subsequently identified as pandemic (H1N1) 2009. As of August 30, over 250,000 cases and 2837 deaths have been documented worldwide (22). This led the WHO to increase the pandemic alert to phase 6 and declare an international public health emergency.

Rapid diagnosis using molecular methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) are the cornerstone to pandemic planning. As outlined in the Canadian Pandemic Influenza Plan (18), provincial Public Health or designate laboratories should have the capacity to identify and subtype influenza viruses using molecular methods. High sensitivity and specificity compared to 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 strains (H1N1 and H3N2), RT-PCR is now being used for influenza A subtyping in order to facilitate the clinical management of patients (8). Influenza A viruses that can not be subtyped must be forwarded to reference laboratories to rule out a novel influenza strain (3, 18). This was the scenario that played out in our and many other laboratories across North America.

On April 24, the Capital District Health Authority (CDHA) microbiology laboratory in Halifax, Nova Scotia received specimens from five symptomatic individuals with epidemiological links to Mexico (4). Three of five were identified as influenza A, but were non- typeable using primers targeting hemagglutinins H1 (huH1) and H3 (huH3) from seasonal influenza A viruses. These three and an additional case were confirmed as pandemic (H1N1) were confirmed by the NML using RT-PCR and sequencing of the M gene (4). While these methods enabled the detection of the first Canadian cases, more timely methods were necessary to help guide public health management. In fact, following confirmation of this novel influenza A virus in Nova Scotia, there was a dramatic increase in the number of respiratory specimens submitted for influenza RT-PCR. Initial strategies based on screening for influenza A followed by subtyping extended turn around times and put tremendous stress on both human resources and available reagents. As such, our traditional testing algorithm had to be quickly revised to accommodate this surge (Figure 1). With primers designed by the NML targeting the HA from H1-lineages of swine influenza A (swH1) and a second primer pair targeting influenza A (5), we validated a duplex RT-PCR for simultaneous detection and confirmation of pandemic (H1N1) 2009.