Rapid typing, subtyping and RNA quantification of influenza virus type A strains in respiratory secretions.

During the winter-spring season 2006-2007, 38 influenza virus strains were identified in patients admitted to hospital with an acute respiratory tract infection. Infections were diagnosed in parallel by direct fluorescent antibody (DFA) staining using type-specific monoclonal antibodies and real-time reverse transcription (RT)-PCR targeting the gene M (nt 25-124). In addition, virus strains were isolated in MDCK cells. Overall, 37 influenza virus strains were type A, and one type B. Of these, 35 (80.4%) were detected and typed by real-time RT-PCR, 34 (80.1%) by DFA, and 27 (71.0%) by virus isolation. Subtyping of 37 influenza virus A strains by RT-PCR and DFA gave the following results: 4/6 H1 strains were correctly subtyped by both methods, while of the 29 H3 strains subtyped by RT-PCR 7 were missed by DFA. Thus, the overall concordance of the two subtyping methods was 28/37 (75.7%). Viral RNA quantification by real-time PCR showed that when respiratory secretion collection was done within 5 days after the onset of symptoms, viral load was greater than 1 x 10(6) RNA copies/ml. In conclusion, typing and subtyping of influenza virus type A strains may benefit from both MAbs and RT-PCR, while viral RNA quantification may provide an indication of symptom onset.