Abstract
In the treatment of influenza, Neuraminidase inhibitors (NAIs) (Oseltamivir and Zanamivir) play a major role. The emergence of variants of influenza A (H1N1) pdm09 virus resistant to Oseltamivir is a matter of great concern as it limits its usage. Therefore, vigilant monitoring for Oseltamivir-resistant viruses has been recommended by the World Health Organization (WHO). Our study aimed to screen the influenza A (H1N1) pdm09 virus for NAI drug resistance during the outbreak of 2015-16 in North-Western India. A total of 640 H1N1pdm09 virus-positive samples were screened for drug resistance to Oseltamivir by WHO allelic discrimination real-time RT-PCR protocol. The allelic discrimination PCR protocol can detect the presence of single nucleotide polymorphisms (SNPs), the H275Y mutation is detected by this method which causes resistance to Oseltamivir. Sanger sequencing of partial fragment of NA gene (fragment IV), of 90 samples were performed to confirm the presence of NA-H275Y mutation. Neuraminidase susceptibility of 20 randomly selected isolates to Oseltamivir was tested using NA inhibition chemiluminiscence based assay. Among 640 H1N1pdm09 positive samples tested, H275Y mutation was detected in one sample (0.15%) by PCR and confirmed by Sanger sequencing also. All the 20 isolates tested for NAI susceptibility by NA star assay were found to be sensitive to Oseltamivir. WHO allelic discrimination PCR is an easy, rapid and sensitive method for high-throughput detection of resistance to Oseltamivir. Systematic regular drug resistance surveillance of Influenza A is essential to monitor the emergence and spread of drug-resistant strains.
Highlights
The first case of Influenza A (H1N1) pdm[09] was reported in Mexico in April 2009 which quickly spread to various countries to cause the pandemic[1]
The emergence of variants of influenza A (H1N1) pdm[09] virus resistant to Oseltamivir is a matter of great concern as it limits its usage
The allelic discrimination PCR protocol can detect the presence of single nucleotide polymorphisms (SNPs), the H275Y mutation is detected by this method which causes resistance to Oseltamivir
Summary
The first case of Influenza A (H1N1) pdm[09] was reported in Mexico in April 2009 which quickly spread to various countries to cause the pandemic[1]. Any mutations that affect this rearrangement may reduce the binding affinity of the drug leading to viral resistance to Oseltamivir[3,4,5]. Emergence of H1N1pdm[09] virus resistant to Oseltamivir is a matter of great concern as it can limit the use of the drug which is the mainstay of treatment of cases infected with H1N1pdm[09]. A point mutation found on the neuraminidase gene, H275Y, is known to cause Oseltamivir resistance. Many methods are available to test for drug resistance to oseltamivir. Sporadic reports of Oseltamivir resistance testing are available from India and data from North Western India is lacking. We tested H1N1pdm[09] positive samples from North Western India for Oseltamivir resistance by allelic discrimination real time PCR to detect for the presence of H275Y mutation. Sanger sequencing and phenotypic assay for drug resistance were done in representative samples
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