HIV-1 drug resistance mutations detection and HIV-1 subtype G report by using next-generation sequencing platform

Highlights

• The preliminary HIV-1 drug resistance by using the NGS method in Iran.

Abstract

Background

Based on world health organization (WHO) recommend, drug resistance assay should be performed in initial of treatment and after treatment for administering and monitoring of anti-retroviral regime in HIV-1 infected patients.

Material and method

NGS analyses were performed on forty-one plasma samples from HIV-1 affected patients using the Sentosa SQ HIV genotyping assay (Vela-Diagnostics, Germany). This system comprises a semi-automated Ion torrent based platform and the sequencing results were analyzed based on ANRS, REGA and Stanford drug resistance algorithms. Phylogenetic analysis was analyzed based on https://comet.lih.lu database as well as MEGA5 Software.

Results

Drug resistances were identified in thirty-three samples (80%) out of forty-one samples. The Phylogenetic analysis results showed that CRF-35AD (94%) and subtypes B (2.4%) and G (2.4%) were dominant subtypes in this study. NRTI and NNRTI associated dominant mutations were M184I/V and K103 N.High-level resistance to lamivudine (3 TC) and Emtricitabine (FTC) were detected in 34.3% of patients while 53.1% were resistant to Efavirenz (EFV) and Nevirapine (NVP). The Protease inhibitor (PI) minor and major mutations were not reported but more than 95% of samples had polymorphisms mutation in K20R, M36I, H69K, L89 M positions. These mutations are subtype dependent and completely are absent in subtype B virus. The secondary mutations were reported in positions of E157Q, S230 N, and T97A of integrase gene and four samples represent low-level resistance to integrase strand transfer inhibitor (INSTI).

Conclusions

This is the first preliminary evaluation of HIV-1 drug resistance mutation (DRM) by using the Sentosa SQ HIV Genotyping Assay in Iran. The NGS represent a promising tool for the accurate detection of DRMs of CRF-35AD that is dominant subtype in Iranian HIV-1 infected population and for the first time revealed HIV-1 subtype G in Iranian population. In the present study polymorphic mutation in the position of K20R, M36I, H69K, L89 M were properly reported in CRF35AD that is dominant in Iranian HIV patients.

Introduction

Human immunodeficiency virus (HIV-1) is responsible for common HIV infections in worldwide [1]. Estimated that more than 37000 people are infected by HIV-1 in Iran [2]. Current treatment for HIV-1 naïve patients consist of a combination of three antiretroviral drugs (ARV) that generally contain nucleoside reverse transcriptase inhibitors (NRTIs), integrase strand transfer inhibitor (INSTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitor (PI) [3]. The viral load is an important factor for monitoring of response and adherence of patients to ARV and should be evaluated in the initiation of treatment and regulated during treatment. However, the aim of ARV treatment is achieving to the constant plasma viral load less than 50copy/ml depends on the used assay [4]. Moreover, the effectiveness of ARV may be compromised by the presence of a mutation in a position of the genome, resulted in drug resistance and virologic failure [5]. The world health organization recommends drug resistance testing in the initial and after treatment failure. Patients with a viral load above 1000coy/ml are the candidate for treatment and virologic failure [6]. The standard genotyping drug resistance testing is involved in protease (PRO) and reverse transcriptase region of the POL gene. Due to the administer of INSTI drugs, it is also recommended to provide drug resistance testing for this class of drugs [3]. Sanger sequencing (SS) usually performed in clinical for detection of the presence of mutation related to drug resistance but SS method enables for detection of mutation presence in 20–30% of the viral population. Recently, Next-generation Sequencing was introduced for evaluating, and analysis of drug resistance in microbiology fields [7]. The sensitivity and reproducibility of this method are better than SS, and this technology enables us to detect and quantify variants with less than 5% of the virus population [8,9]. Recently, many studies compared and evaluated the results of drug resistant mutation (DRM) by using NGS and SS. They demonstrated and confirmed that the NGS method detects all related drug resistance mutations found by SS besides the identification of additional mutations [[10], [11], [12]]. The Sentosa SQ HIV genotyping assay (Vela-Diagnostics, Germany) is invitro and Semi-automated with novel NGS method for evaluation and analysis of HIV-1 drug resistance mutation. The objective of this study was a monitoring of drug resistance mutations in HIV-1 infected patients, who are candidates of treatment failure, by using NGS method for the first time in Iran.