High resolution melting analysis as a tool to detect molecular markers of antiviral resistance in influenza A viruses.

A real-time PCR followed by high resolution melting analysis (HRMA) was developed, for rapid detection of antiviral resistance markers in influenza A viruses, of both H1N1 and H3N2 subtypes. The targets of these assays were the nucleotide substitution G806A (S31N mutation) in the M gene as marker of resistance to adamatanes in influenza viruses A(H3N2), the substitution A356T (E119V mutation) in the N2 gene of influenza viruses A(H3N2) and the substitution C823T (H274Y mutation) in the N1 gene of the pandemic A(H1N1) 2009 virus as markers of oseltamivir resistance. First, the designed primers and the overall protocol of the HRMA were validated using already characterized viral isolates either containing or lacking changes at the tested codons. Then, HRMA was used to search for the marker of oseltamivir resistance in 75 clinical samples, H1N1 2009 positives, analyzed previously by pyrosequencing and Sanger sequencing, and of both adamantane-derivatives and oseltamivir resistance in 57 H3N2 positive clinical samples. The results of HRMA of the H1N1 2009 isolates were in agreement with those obtained by sequencing. As regards the H3N2 isolates, HRMA revealed a widespread resistance to adamantanes with 89.5% nucleotide substitution G806A, while 3% were resistant to oseltamivir (A356T change). HRMA, applied to the detection of markers of resistance to antiviral drugs against influenza A viruses, confirmed to be a procedure flexible, low cost and time-saving, suitable for application to epidemiological surveys and in clinical settings for diagnostic purposes.