Dual infection with influenza A and B viruses

A throat swab from a 7-year-old child with a mild influenza-like illness was cultured for respiratory viruses by standard and rapid plate culture methods. Standard culture yielded influenza B, whereas influenza A was obtained from rapid plate culture. Testing of the original sample by PCR confirmed the presence of both viruses. A surveillance program (Viral Watch) for respiratory viruses showed cocirculation of both these viruses in the community during this period. Influenza A epidemics occur yearly in temperate zones with seasonal regularity. There are some seasons where there may be cocirculation of influenza B, which may appear as separate peaks of respiratory-like illness in the epidemiologic data. In most cases it is difficult to distinguish between these two agents on clinical symptoms alone, because the individual response is variable, as is the degree of illness associated with the different influenza A and B subtypes. Dual infections with other respiratory viruses have been estimated to occur in fewer than 5% of the total numbers of cases. 1 Mixed infections usually include respiratory syncytial virus and adenovirus, or influenza A or B with one of the parainfluenza viruses. 1–4 In Alberta there has been a community-based surveillance program (Viral Watch) since 1976 which monitors influenza-like illness in the community, especially during the months of September to March, when significant numbers of influenza-like illnesses occur. The Viral Watch program is based on sentinel family physician reports, geographically distributed in the province, tracking respiratory illness through data collection and also taking swabs for viral culture from patients with a respiratory illness. The swabs are sent to the Provincial Laboratory for culture for various viral respiratory agents. The numbers of virus isolates and patients with respiratory disease are collated, published in a timely manner and disseminated to national, provincial and regional public health authorities. Case report and methods. A 7-year-old girl visited her family physician in December with a mild respiratory illness. Her physician collected a throat swab into M4 Viral Transport Medium (Dalynn Biologicals, Calgary, Alberta, Canada) for viral culture, in keeping with the protocol of the Viral Watch Program. In this laboratory two methods are used to detect respiratory viruses, a rapid culture with the use of a mixed cell assay and standard culture. For rapid culture 0.2 ml of the sample is inoculated into a microwell plate containing a monolayer of the two cell lines (mink lung and NCI-H292; BioWhittaker, Walkersville, MD), centrifuged and incubated overnight. The next day the cells are scraped, spotted onto a glass slide and stained by fluorescent-tagged polyclonal reagents (Dako Diagnostics Ltd., Ely, UK) to influenza A and B, respiratory syncytial virus and parainfluenzas 1, 2 and 3. Samples that screen positive are retested to ascertain the specific virus by fluorescent-tagged monoclonal specific stains. Standard culture is performed by inoculating 0.2 ml of sample into rhesus monkey kidney cells (BioWhittaker), primarily for the influenza and parainfluenza agents, and A549 for other agents, especially adenovirus. The rhesus monkey kidney tubes are incubated at 33°C on a roller drum, and twice weekly hemadsorbing agents are detected by replacing the culture fluid with a 0.1% suspension of human O cells. If the culture is positive the supernatant is titrated to determine the titer of the agent (previously identified in rapid culture) for subtyping by the hemagglutination inhibition assay, with antisera prepared against strains in the current year’s influenza vaccine. Results. In this case the sample was determined to be positive for influenza A by rapid culture after overnight incubation, and a few days later a hemadsorbing agent was detected in standard culture. However, the hemadsorbing agent could not be subtyped to one of the influenza A strains, based on the rapid culture result, and was sent to the National Microbiology Laboratories for investigation of an unusual subtype. (In previous experience the influenza isolates from rapid and standard cultures have always matched.) The sample was cultured again, and on the second occasion the hemadsorbing agent was tested for its identity. The National Microbiology Laboratories and ourselves found this isolate to be influenza B; hence the original sample was sent to the National Microbiology Laboratories for molecular characterization by reverse transcription-PCR to influenza A and influenza B, 5 both of which were detected in the sample. Discussion. There have been no published reports of influenza A and B coinfection in humans, although these have been inferred through serologic investigations of various groups. 6 This particular season both influenza viruses were cocirculating in the community, and this combination of events may have made a dual infection possible. A national survey of the influenza subtypes in circulation showed that A/New Caledonia/20/99-like (H1N1) predominated with a few A/Panama/2007/99-like (H3N2) strains, together with B/Yamanashi/166/98-like. At the time of this patient’s exposure the numbers and ratios of influenza A and B isolates obtained in culture locally were similar based on the laboratory isolation data from respiratory samples submitted from the Viral Watch physicians. In addition we found that the influenza A isolate outgrew the influenza B in the rapid culture assay, whereas the reverse occurred in standard culture, thus allowing for the identification of both agents. In vitro studies of mixed influenza infections with the vaccine and wild-type strains showed little or no inhibition of either strain, depending on the infectious dose, the host and time of assay. 7 The patient’s clinical presentation was a mild manifestation of an influenza-like illness, from which she recovered without sequelae. Influenza A infections are generally more severe than influenza B illnesses depending on the infecting subtypes. In immunocompetent patients dual infections do not cause more severe disease 1 than a single agent, but the same may not be true for those who have underlying immunodeficiencies. 1 Our patient had no known underlying immunodeficiencies. Acknowledgments. We thank the technologists of the Virology Department of the Provincial Laboratory for their technical assistance and expertise.

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