Detection of oseltamivir sensitive/resistant strains of pandemic influenza A virus (H1N1) from patients admitted to hospitals in Thailand

Oseltamivir has been used widely for prophylaxis or treatment during outbreaks of the pandemic influenza virus (H1N1) in several countries. The aim of this study was to develop a real-time RT-PCR (reverse transcription-polymerase chain reaction) to be applied for detection and monitoring of the oseltamivir resistant strains of this virus during three outbreaks (May 2009 to October 2010) in Thailand. The real-time RT-PCR assay for detecting H275Y proved highly specific for the pandemic influenza virus (H1N1) as no cross-amplification was detected with other respiratory viruses or human total RNA. The assay was also highly sensitive with a detection limit as low as 100 copies/μL for both wild-type and resistant strains. The performance of the assay was evaluated in terms of amplification efficiency (100%). The results obtained by real-time RT-PCR were in complete agreement with direct nucleotide sequencing. However, real-time RT-PCR provided more detail on the relative quantities of ratios between resistant and sensitive strains in each individual. The results revealed that four of 1288 (0.31%) patients were infected with the oseltamivir resistant strain. The number of patients infected by resistant strains was higher during the third (0.61%) and second (0.24%) waves than during the first (0%) outbreak. In conclusion, the real-time RT-PCR assay for H275Y detection is advantageous because it is specific, sensitive, and provides quantitative data. And it would be useful for large-scale testing and monitoring of oseltamivir resistant strains of the pandemic influenza A virus (H1N1).

► We developed the method for detecting oseltamivir resistant strain of pH1N1.
► The method used real time PCR to detect H275Y mutant.
► The assay is highly sensitive with a detection limit as low as 100 copies/μL.
► The number of patients infected by resistant strains increased with time.
► This method is advantageous for monitoring of pH1N1 oseltamivir resistant strains.