Pandemic (H1N1) 2009 and Seasonal Influenza A (H1N1) Co-infection, New Zealand, 2009

Co-infection with seasonal influenza A (H1N1) and pandemic (H1N1) 2009 could result in reassortant viruses that may acquire new characteristics of transmission, virulence, and oseltamivir susceptibility. Results from oseltamivir-sensitivity testing on viral culture suggested the possibility of co-infections with oseltamivir-resistant (seasonal A [H1N1]) and -susceptible (pandemic [H1N1] 2009) viruses.


The Study
Infl uenza viruses were identifi ed through the New Zealand national infl uenza surveillance system as part of the World Health Organization global program for infl uenza surveillance previously reported (2). Pandemic (H1N1) 2009 virus dramatically increased demand for infl uenza subtyping (2), necessitating a change in the standard real-time reverse transcription-PCR (rRT-PCR) algorithm. Samples were fi rst screened with singleplex universal infl uenza A and pandemic (H1N1) 2009 assays (5,6). If negative results were obtained for both of these tests, samples were then tested for infl uenza B. If samples were positive for universal infl uenza A but not for pandemic (H1N1) 2009 virus, they were subtyped for seasonal H1 and H3 by rRT-PCR. Samples positive for pandemic (H1N1) 2009 virus were not subsequently assayed for other infl uenza viruses during testing but were tested at the end of the Southern Hemisphere infl uenza season as part of this study.
After the discovery of co-infection in viral culture, 1,044 clinical samples that were positive for pandemic (H1N1) 2009 were screened by rRT-PCR for seasonal A (H1N1) virus. Eleven co-infections were identifi ed. Two additional samples indicated co-infections when viral culture was screened by rRT-PCR but could not be confi rmed because our laboratory did not receive the original clinical specimen.
Laboratory contamination of viral culture could account for the presence of both infl uenza subtypes in viral culture samples. Co-infection was confi rmed by using World Health Organization-recommended specifi c singleplex rRT-PCRs (5) on each of the 11 original clinical specimens (Table). The specifi c rRT-PCRs each are specifi c for the gene segment encoding hemagglutinin; 1 assay is specifi c for pandemic (H1N1) 2009, the other for seasonal infl uenza A (H1N1). The 2 assays were run in parallel for each sample with appropriate controls, including specifi city controls.
Within this small number of cases, 10 of the 13 patients were female, and 6 patients were of Maori descent. Each fi gure was higher than the expected 51% and 14.6% representation in the New Zealand population, respective- ly, but co-infections were too few to draw any conclusions based on these characteristics (Table).
A vaccine for pandemic (H1N1) 2009 was not available when these samples were collected (June-November 2009), and only 1 of the 13 patients had a history of seasonal infl uenza vaccination. None of the 13 case-patients had severe illness or were hospitalized.
Eight of the 13 case-patients came from the central North Island; the remainder came from Auckland (2), Wellington (2), and Otago (1). All of these regions had high infl uenza activity during the 2009 New Zealand infl uenza season (2).
For each case, details of initial and ongoing transmission were unclear. Two cases occurred in a husband and wife, who had onset of symptoms on the same day; the remaining cases are not thought to be linked. All cases were reported after pandemic (H1N1) 2009 had become widespread in the community; therefore, contact tracing had ceased within New Zealand.
Dates of illness onset for all case-patients occurred within a 16-day period (June 14-30). This period coincided with the short period when both seasonal A (H1N1) and pandemic (H1N1) 2009 viruses cocirculated at approximately equal levels in the community, before the pandemic virus became the predominant strain (2) (Figure).

Conclusions
Results from oseltamivir-sensitivity testing on viral culture suggested the possibility of co-infections in patients with both resistant (seasonal A [H1N1]) and susceptible (pandemic [H1N1] 2009) viruses. This test required both viruses to grow suffi ciently in cell culture and grow to similar titers. Only by this approach was co-infection discovered and later investigated by use of more sensitive and highly specifi c rRT-PCRs.
Co-infections of different infl uenza viruses are rarely reported; reports focus solely on co-infections of infl uenza A and B, not of 2 infl uenza A subtypes (8)(9)(10)(11). Two recent studies, 1 examining 2,273 clinical infl uenza samples with multiplex PCR methods found no infl uenza co-infections (12,13); another study estimated infl uenza co-infections to be as high as 3% (14). The rate of co-infection determined in this study was 1.1% (n = 1,044), which may underestimate the actual rate because not all tests used in this study (either biochemical or molecular) screened for co-infection  Although infl uenza co-infections are rare, we have shown that they occurred during the fi rst stage of a pandemic when seasonal strains cocirculated. This cocirculation poses a risk for further reassortment for the pandemic strain, which could result in a new pandemic strain. Of particular concern is the potential generation of an oseltamivir-resistant pandemic strain. The genesis of a harmful infl uenza reassortant warrants further investigation in animal models or in vitro systems. Further analysis of natural co-infections may help elucidate a role for the human host in infl uenza reassortment.