1 INTRODUCTION

The influenza virus causes mild to severe respiratory infections resulting in 290 000−650 000 deaths globally.¹ The Influenza viruses are spherical, filamentous, or pleomorphic with a diameter of ~100 nm. They have a lipid envelope with characteristic spikes called haemagglutinin and neuraminidase (NA).² The fundamental activity of the NA protein involves cleaving of the sialic acids on the cell surface that cause the release of virions and infection of new cells.³ Oseltamivir carboxylate mimics the natural substrate of NA (sialic acid) and interacts with the active site, making Glu276 to rotate and bond with Arg224 which is stabilized by His275. These conformational alterations prevent the binding of sialic acid, and the subsequent release of the virus.⁴ The active sites of NA are known to be conserved, although mutations can affect these sites which may change virus transmissibility as well as drug sensitivity.^{5, 6} A point mutation in NA gene, H275Y, which is due to a nucleotide change of Cysteine (C) to Thymine (T) at 823 position resulting in an amino acid change of Histidine (H) to Tyrosine (Y) at 275 position conferring oseltamivir resistance is one of the most significant mutations.^{5, 7, 8}

Antiviral usage has increased in recent times due to its effectiveness in reducing infections resulting in mild to moderate levels of resistance. Global Influenza Surveillance and Response System (GISRS) has deployed surveillance for influenza antiviral susceptibility testing. The GISRS states that phenotypic and genotypic assays are available to determine if an influenza virus is susceptible to NAIs. Genotypic assays comprise single-nucleotide polymorphism (SNP) detection, gene sequencing, and pyrosequencing.⁹ According to World Health Organization (WHO), the SNP based assay is currently used only for detection of H275Y mutation in influenza A(H1N1)pdm09 viruses and has been used in similar studies.^10-14

Resistance to oseltamivir for A/(H1N1)pdm09 started to appear and was reported to affect immunocompromised individuals¹⁵ as well as patients who were treated with the drug.⁵ Resistance to oseltamivir has been detected as 2.14% in China,¹⁶ 1.3% in Japan,¹⁷ 1.2% in United States,¹⁸ 1% in United Kingdom,¹⁵ and 0.74% in Indonesia.¹⁰ According to a study conducted by Trivedi et al.¹² in India, the prevalence rate was 0.15% while Saha et al.¹⁹ and Nandhini et al.²⁰ found that all the samples were sensitive to oseltamivir. In India, studies on the prevalence of oseltamivir resistant Influenza A(H1N1)pdm09 viruses are limited especially after the year 2015. Further, number of samples used in the previous studies are less and hence the possibility of detection of resistant virus is low. Thus, this study aims to detect the presence of H275Y mutation among Influenza A(H1N1)pdm09 patients from 2014 to 2021.

2 MATERIALS AND METHODS

3 RESULTS

3.2 Allelic discrimination real-time RT-PCR

Endpoint fluorescent signals were analyzed and the threshold was adjusted manually. The results were categorized as homozygous wild-type (H275), homozygous mutant-type (Y275), and heterozygous type (H275/Y275).

Out of the 151 pools that were tested for H275Y mutation, 140 pools were homozygous H275 and only 1 pool showed amplification for both H275 and Y275 (heterozygous type) and 10 pools were negative for both H275 and Y275 (Figure 1 and Table 1).

The five samples of the heterozygous positive pool were tested individually and one sample was detected as homozygous mutant-type (Y275). The prevalence rate of oseltamivir resistance from 2014 to 2019 was found to be 0.13%.

3.3 Modified Sanger sequencing

Sequence analysis of homozygous Y275 sample in comparison to reference strains showed a nucleotide change of CAC to TAC at 823 position resulting in an amino acid change from H to Y at 275 position, confirming the presence of H275Y mutation.

Fifty samples from the 10 undetermined pools of 2020 and 2021 were sequenced and sequence analysis showed no nucleotide change at 823 position for 49 samples, however one sample from 2020 showed nucleotide change of CAC to TAC at 823 position resulting in an amino acid change from H to Y at 275 position. The prevalence rate of oseltamivir resistance from 2014 to 2021 was found to be 0.27%. All the nucleotide sequences (510 bp) from this study were deposited in GenBank with accession numbers from OP737518 to OP737538 and OQ363464 to OQ363489. Among these, OP737518 and OQ363483 had the codon change of CAC to TAC with amino acid mutation of H to Y.

Sequence alignment of all 2020 and 2021 samples with the reference sequences and WHO recommended probe showed a mutation of GAG to GAA at 831 position. This nucleotide mismatch with probe could have possibly resulted in negative results for both H275 and Y275 targets in allelic discrimination PCR.

Further, nucleotide BLAST analysis of the probe binding region using GISAID analysis tool showed that the nucleotide change of G to A at 831 position has been reported since end of 2019 (GISAID No. EPI_ISL_16071680) in different regions of the world and this nucleotide change is also observed in the WHO recommended vaccine strain for 2021−2022—Northern hemisphere (Figure 2).

4 DISCUSSION

Oseltamivir is the most widely used antiviral for the treatment of influenza infections in India, therefore it is important to evaluate its susceptibility and effectiveness. H275Y mutation leading to oseltamivir resistance has a prevalence of 1%−2% worldwide and less than 1% in India. Gohil et al.²² conducted a study for the surveillance of the influenza virus in Mumbai and found that all the samples were sensitive to oseltamivir. Similar studies by Sahu et al.¹³ during 2009−2015 and Trivedi et al.¹² during 2015−2016 demonstrate the prevalence of oseltamivir resistance as 1.4% and 0.15%, respectively.

In India, the number of published studies on oseltamivir resistance after the year 2015 are sparse. Hence, this study was conducted to estimate the prevalence of oseltamivir resistance among Influenza A(H1N1)pdm09 patients from 2014 to 2021 using allelic discrimination real-time RT-PCR to identify H275Y gene mutation.

The current study, estimates prevalence rate of oseltamivir resistance as 0.27% among hospitalized patients that was found to be similar to previous studies from India.^{12-14, 19, 20} Based on the results, it can be concluded that oseltamivir resistance is still less than 1% in India. Out of two resistant samples, one was from the year 2017, collected from 26-year-old male, with 2 days history of fever, cough, coryza, red eye, retro-orbital pain, myalgia, and mild gastrointestinal symptoms. He had received oseltamivir treatment and there was no history of previous influenza infection or oseltamivir treatment. However, the follow-up report showed the patient had recovered and was healthy. The second resistant sample (collected in 2020) was from a 55-year old male, with 2 days history of fever, cough, difficulty in swallowing, breathlessness, and gastrointestinal symptoms. The second patient had also received oseltamivir treatment with no previous history of infection or resistance. However, the patient's condition deteriorated and hypoxia worsened, secondary bacterial infection was also detected. Due to the patient's financial restraints he was discharged against medical advice. A few studies from Hong Kong,²³ Vietnam,²⁴ and United States,¹⁸ have reported oseltamivir resistance in patients without prior history of treatment. A similar finding was reported by Tandel et al.²⁵ in a study conducted in Madhya Pradesh, India, where drug resistance was detected in patients without any previous exposure. Oseltamivir resistant and oseltamivir-susceptible viral infections are reported to be clinically similar, with the exception of those who are immunocompromised or have comorbid conditions.²⁶

Allelic discrimination real-time RT-PCR is a rapid SNP detection method that detects H275Y mutation and has been recommended by WHO. In the current study, pooled sample testing strategy was used to detect H275Y gene mutation. Pooled sample testing strategy has been used to detect viruses such as hepatitis B virus, human immunodeficiency virus, influenza virus among others.²⁷ This testing strategy with pool sizes of 5−10 samples has also been widely used to increase testing capacity and conserve resources during upsurge testing periods such as the on-going COVID-19 pandemic.²⁸ A study by Shukla et al. showed that the average change in C_t for pool of 5 was +2.56 and +3.38 for pool of 10.²⁸ To avoid false positives and increase efficiency of allelic discrimination real-time RT-PCR, pools of 5 influenza A(H1N1)pdm09 positive samples with C_t <30 were tested for SNP detection.

In this study, the samples of 2020 and 2021 were negative for both wild-type and mutant-type. Based on sequencing results, it was confirmed that 49 samples did not have the H275Y mutation, but one sample was detected with H275Y mutation. On further analysis of sequences, an additional nucleotide mutation was detected at the probe binding site that might have contributed to negative results for both H275 and Y275. Allelic discrimination assays are very specific and a single mismatch between the template and the probe sequence will affect its hybridization.²⁹

Further, nucleotide BLAST analysis of probe binding region showed that these mutations might have emerged toward the end of 2019 (GISAID No. EPI_ISL_16071680, unpublished data) and has been detected in many countries since its emergence. The WHO recommended vaccine strains sequence for Northern Hemisphere—2021−2022 also documents this change (GISAID No. EPI_ISL_404527). Although, the nucleotide change at 831 position results in a synonymous mutation and does not affect drug binding, the mismatch at the probe binding site is a major concern since these probes are recommended by WHO for detection of the substitution at amino acid 275 in the NA of influenza A(H1N1)pdm09 viruses. The WHO, allelic discrimination real-time RT-PCR is considered as a rapid and convenient method and is widely used for screening of mutations associated with drug resistance.¹² The detection of nucleotide change in probe binding site of 2020 and 2021 samples strengthens the fact that considering the high mutation rates among influenza A viruses the WHO protocol recommendations needs to be revised periodically to avoid false results.

5 CONCLUSION

The evolving nature of influenza viruses poses a challenge for developing vaccines and antivirals. The NA inhibitors against influenza viruses are effective and used extensively. Any mutation in the drug's binding site could result in a decrease in the drug's effectiveness and eventual discontinuation. This study illustrates a 7-year profile of H275Y gene mutation leading to oseltamivir resistance among Influenza A(H1N1)pdm09 patients from 2014 to 2021. The result shows a lower prevalence of 0.27% which indicates that oseltamivir carboxylate can be continued to be used as a therapeutic. The negative results obtained in the samples of 2020 and 2021 using allelic discrimination real-time RT-PCR following WHO protocol highlights the necessity of monitoring the mutations in the influenza virus and implementing it in the respective assays so as to avoid any false negative results.

AUTHOR CONTRIBUTIONS

Anitha Jagadesh conceived, and guided the research work. Smriti Krishna performed most of the experiments. Anup Jayaram contributed to data processing and analysis. Ujwal Shetty contributed to performing sequencing. Prasad Varamballi contributed in data analysis. Chiranjay Mukhopadhyay provided insightful discussion. All authors have read and approved the manuscript

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

The study was reviewed and approved by the Institutional Ethical Committee (IEC), Kasturba Medical College, and Kasturba Hospital (Approval ID-IEC2: 179/2022).