The early detection of infected patients, the implementation of isolation measures, and contact tracing are imperative for the management of influenza virus infections. Rapid antigen tests can generate a result in 30 min or less (18
); unfortunately, these methods lack sensitivity (Table ) compared to that of RT-PCR (7
) and should not be used to exclude the possibility of an influenza virus infection. Early in the pandemic (H1N1) 2009 influenza virus outbreak, the only available guidelines suggested that the identities of novel influenza viruses should be confirmed by viral culture and at least partial sequencing of the viral genome (3
). While sequencing is considered the gold standard for the confirmatory identification of novel influenza viruses, the use of this approach is impractical for most laboratories and poses problems for the routine detection of influenza viruses by RT-PCR (discussed later). Using sequence data from pandemic (H1N1) 2009 influenza virus, we designed primers targeting swH1 and validated the performance of a conventional RT-PCR assay that is capable of simultaneously detecting and confirming the identity of this novel influenza A virus.
The duplex RT-PCR assay was significantly more sensitive than all other assays for the detection of influenza A virus (Table ), including the conventional triplex RT-PCR assay previously used in the CHDA microbiology laboratory (Fig. ). This prompted a rapid modification of the influenza testing algorithm during the Nova Scotia outbreak (Fig. ). Interestingly, the duplex RT-PCR assay was also more sensitive than the real-time influenza A virus RT-PCR designed by the CDC (2
). The lower sensitivity of the real-time RT-PCR could partly be attributed to the protocol, which had not yet been optimized for use on the LightCycler platform. However, Poon et al. (17
) also recently found that a real-time RT-PCR assay was less sensitive than a conventional RT-PCR assay for the detection of A/Swine/Hong Kong/PHK1578/03 virus. It remains to be determined whether the sensitivity of real-time influenza A virus RT-PCR assays could be enhanced by using other platforms or a modified protocol.
Several conclusions could be derived by comparing the results obtained with the monoplex and the duplex RT-PCR assays. Even though identical primers were used in both assays, the duplex RT-PCR assay was significantly (P
< 0.001) more sensitive (Fig. and Table ). Several possibilities could explain these discrepant results. First, the two assays differed in respect to the extraction methods, RT-PCR conditions, and thermocycling conditions (annealing temperatures) used. For example, the higher annealing temperature used in the duplex RT-PCR assay (55°C) compared to that used in the swH1 monoplex RT-PCR assay (50°C) contributed to increased sensitivity (Fig. and Table ). Second, some of the discrepant results may simply reflect a Poisson distribution due to sampling error with low concentrations of template (11
). This phenomenon is almost impossible to control and is the most pronounced with low target concentrations, in which small changes in the amount of the nucleic acid template in a PCR mixture could generate a relatively large difference in the numbers of amplicons produced. A large number of replicates would be necessary to overcome this limitation. Similarly, PCR inhibitors are known to affect PCR amplification and could lead to considerable variations in the efficiency of the PCR. This hypothesis is highly plausible, since the monoplex and the duplex RT-PCR assays were performed by using different extraction methods; however, the lack of an exogenous internal control prevents assessment of the contribution of PCR inhibitors. Further studies are being undertaken to combine the duplex RT-PCR assay with the detection of an internal control, such as bacteriophage MS2.
In addition to identifying influenza A virus, the duplex RT-PCR assay was highly sensitive at identifying swH1 (Table ). During the validation period and retrospective analysis, 94% (62/66) and 92% (34/37) of the cases of pandemic (H1N1) 2009 influenza virus were found to be swH1 positive by the duplex RT-PCR assay. It should be noted that the annealing temperature of the duplex RT-PCR assay greatly influenced the ability of the assay to detect swH1 (Fig. and Table ). The use of an annealing temperature of 55°C rather than one of 50°C increased the number of swH1 targets detected (Table ). Using this strategy, the duplex RT-PCR assay identified swH1 in the majority of cases of pandemic (H1N1) 2009 influenza virus, thereby considerably reducing the number of specimens forwarded to reference laboratories for confirmatory identification. However, we recognize there will be circumstances in which influenza A virus-positive specimens will have virus at amounts below the limit of detection of the swH1 primer pair. For the few cases that fit this criterion, low viral loads were observed by the real-time RT-PCR assay. Since the sensitivities of RT-PCR assays targeting influenza A virus are not equivalent (Table ), confirmatory identification of pandemic (H1N1) 2009 influenza virus could be problematic when specimens contain low viral loads. The use of sequencing has been proposed by some to confirm the detection of pandemic (H1N1) 2009 influenza virus in clinical specimens; however, this methodology can also be problematic. Sequencing reactions use the amplicon generated from the RT-PCR assay as the template; therefore, amplicon contamination could result in false-positive sequence data. With the recognition that sequencing will always play an important role in the confirmatory identification of novel influenza viruses, other methods for the confirmatory identification of low-positive RT-PCR results should be sought (discussed below).
Since the RT-PCR assay optimal for the detection of pandemic (H1N1) 2009 influenza virus is unknown, a modified gold standard was used in this study to assess the clinical performance of all RT-PCR assays. A positive case was defined by the detection of concordant results between at least two RT-PCRs targeting different genomic regions and subsequent sequence analysis to ensure the specificities of the primers. As some discordant results between the various RT-PCR assays were observed, other experiments were required to ensure that the additional influenza A virus-positive cases detected could not be attributed to amplicon contamination. Specimens displaying discordant results were subjected to reextraction and repeat RT-PCR by the same duplex assay, a real-time swH1 RT-PCR assay, and reextraction by use of a larger volume of specimen (1 ml versus 140 μl) and a second RT-PCR with primers whose sequences were outside the original targeted region, followed by sequencing of the M gene. Using these strategies, all discordant results could be resolved. In light of all results, the duplex RT-PCR assay was deemed to be highly sensitive for the detection of influenza A virus and the confirmatory identification of pandemic (H1N1) 2009 influenza virus.
Until now, most infections attributed to pandemic (H1N1) 2009 influenza virus have been mild and self-limited. There is growing concern that this virus will evolve and lead to subsequent outbreaks of severe disease. Rapid detection of this novel influenza virus is paramount so that control measures may be implemented. We have shown that the duplex RT-PCR assay is a highly sensitive, accurate, and reliable method for the detection and confirmatory identification of pandemic (H1N1) 2009 influenza virus. While the real-time RT-PCR assay could permit a more timely diagnosis (12
), not all laboratories have the infrastructure to offer such testing. The duplex RT-PCR assay is undoubtedly an attractive option for laboratories without this capability. Since the fate of this novel influenza virus is unclear, a conventional RT-PCR assay that uses generic reagents (without the need for probes or specialized kits) is ideal for any laboratory transitioning from low- to high-throughput screening for pandemic (H1N1) 2009 influenza virus. With generic reagents, supplies may also be more readily accessible as the global demand for testing peaks.