In recent years, influenza type A has provided a good example of the challenges confronting real-time PCR detection of viral agents comprising genetically heterogeneous populations. Influenza A exists as multiple subtypes showing considerable sequence variation between and within subtypes. However, the problems concerning the PCR detection of influenza A are not due to a lack of available sequence information. Genbank, for example, contains an extensive amount of sequence data for numerous genes from a wide range of viral subtypes representing various regions around the world.
Rather, the recent problems concerning PCR detection of influenza A have involved a change in the genetic nature of the viral subtypes circulating in a population. Following the Hong Kong pandemic of 1968, the influenza A types infecting humans have remained fairly stable with the common subtypes causing disease including H1N1, H1N2, H2N2 and H3N2. As a consequence, numerous PCR assays have been developed to ensure the detection of these subtypes. However, in the late 1990s avian strains of influenza were shown to infect humans (Hammel and Chiang, 2005), and several incidents of infection were reported, including H5N1, H7N2, H7N3, H7N7, and H9N2, in countries including Canada, the United States, the Netherlands as well as numerous Asian countries. Established PCR protocols were unable to detect these avian strains due to the genetic heterogeneity of their genomes, and, consequently, there was a growing need to review diagnostic capabilities for influenza A in many countries.
These limitations of the established PCR protocols were addressed by the development of a conventional PCR assay capable of detecting influenza A viruses from multiple animal species (Fouchier et al., 2000). More recently, our laboratory developed a 5' nuclease real-time RT-PCR (WhSl-FluA-5N; protocol 1) for the detection of both the common human influenza A subtypes as well as the avian subtypes now known to infect humans. To validate our WhSl-FluA-5N assay, it was compared to a previously described influenza A 5' nuclease RT-PCR assay (FluA-TM; van Elden et al., 2001), which also targeted the influenza A matrix protein gene. Overall, the results showed good agreement between the two assays when applied to the detection of influenza type A in nasopharyngeal aspirate samples obtained from patients in our local Australian population. However, the WhSl-FluA-5N assay could detect a greater number of influenza A subtypes than the FluA-TM.
Briefly, nucleic acids were extracted from 23 influenza type A cultures comprising subtypes H1N1 (6 strains), H3N2 (12), H5N1 (2), H5N3 (1), H7N7 (1), and H9N2 (1), and were tested by both assays. All H1N1 and H3N2 strains were detected by both assays, whereas, the H5N1, H5N3, H7N7, and H9N2 strains were positive by the WhSl-FluA-5N assay only (Whiley and Sloots, submitted for publication). It should be noted that the FluA-TM assay has been used successfully in our laboratory for the detection of influenza A for a number of years, and only recently have we adopted the WhSl-FluA-5N assay given the potential introduction of avian strains into our population.
On the whole, the above results highlight that the range of influenza A subtypes affecting humans can change and that, given the sequence variation existing between such subtypes, laboratory diagnostic assays need to be reviewed as new subtypes infecting humans are identified. Potentially, the use of the WhSl-FluA-5N assay may need to be reassessed if other influenza A subtypes are identified or if genomic sequence variation of local strains leads to mismatches in the primer or probe target sites.
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