Abstract
In India, the roll out of the free antiretroviral therapy (ART) program completed a decade of its initiation in 2014. The success of first-line ART is influenced by prevalence of HIV pretreatment drug resistance (PDR) in the population. In this cross-sectional study, we sought to determine the prevalence of PDR among adults attending the state-sponsored free ART clinic in Pune in western India. Fifty-two individuals eligible for ART as per national guidelines with median CD4 cell count of 253 cells/mm3 (inter quartile range: 149–326) were recruited between January 2014 and April 2015. Population-based sequencing of partial pol gene sequences from plasma specimen revealed predominant HIV-1 subtype C infection (96.15%) and presence of single-drug resistance mutations against non-nucleoside reverse transcriptase inhibitor in two sequences. The study supports the need for periodic surveillance, when offering PDR testing at individual level is not feasible.
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ART-naive individuals eligible for ART as per national guidelines were recruited voluntarily between January 2014 and April 2015. The other criteria for inclusion were age more than 18 years, documented HIV-1 infection, and absence of prior exposure to any antiretroviral (ARV) drugs except for a single dose of nevirapine during pregnancy for prevention of mother to child transmission. The study was approved by the ethics committee of the National AIDS Research Institute. Ten milliliters of blood was collected from each participant for CD4+ cell count, viral load estimation, and drug-resistant genotyping after obtaining written informed consent. The demographic and clinical details were recorded from the data available at the ART clinic. Prior exposure to ARV was ascertained by personal interview at the time of pre-ART counseling. Patients' characteristics were summarized by medians and interquartile range (IQR) for continuous variables and proportions for categorical variables.
Genotyping to characterize drug resistance mutations (DRMs) was performed at the WHO accredited laboratory of the National AIDS Research Institute (Pune) using an in-house population-based sequencing method, validated previously. 5 Complete protease (1–99 amino acids) and partial reverse transcriptase (1–256 amino acids) of the pol gene were sequenced on 3130xl DNA genetic analyzer (Applied Biosystems, Foster City, CA). Estimation of PDR prevalence was performed by a calibrated population resistance (CPR) tool (version 6.0) available at Stanford HIV drug resistance database. 6
A total of 52 ART-naive individuals with median age of 37.5 years (IQR: 34–42) participated in the study. The clinical and demographic characteristics of these subjects are summarized in Table 1. The predominant mode of transmission was heterosexual and 62% of them were female. The median CD4 cell count and plasma viral load were 252 cells/mm3 (IQR: 149–326) and 59,179 copies/ml (IQR: 21,381–177,097), respectively. All the 52 plasma samples were successfully genotyped. The Phylogenetic tree was generated using PhyML interface with the maximum likelihood method under the General Time Reversible model.
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It showed clustering of 50 sequences with HIV-1 subtype C pol gene reference sequence (Fig. 1). A solitary sequence (NARI-SK_N_14–916) clustered with subtype A1 and another sequence (NARI-SK_N_11–35934) were segregated away from subtype C cluster, which showed features of subtype A1/C recombination on analysis with Recombinant Identification Program (
The phylogenetic tree based on partial pol gene fragment of HIV-1. The relationship between partial pol gene sequences from 52 antiretroviral therapy-naive individuals and reference sequences retrieved from the Los Alamos National Laboratory HIV Sequence Database is shown.
IQR, interquartile range; MSM, men who have sex with men.
In resource-limited settings, when offering drug resistance genotypic assay to all individuals eligible for ART is not feasible, periodic monitoring of the population for emergence of drug resistance is vital. Surveillance of population-level resistance can be divided into three main categories: transmitted drug resistance (TDR), PDR, and acquired drug resistance (ADR). Although multiple reports on TDR and ADR are available from India, there is limited data available on PDR. A prior study among individuals eligible for ART from Mumbai (India) was conducted in 2008 and reported 8% prevalence of NNRTI-associated DRMs. 8 Incidentally, there were no NRTI-associated DRMs, similar to our study. PDR may arise because of prior exposure to ARV or it may be present exactly at the time of infection (TDR). The two NNRTI-associated SDRMs encountered in this study were V106M and K103N. The individual harboring V106M is a polygamous 48-year-old male, who reportedly acquired infection by the heterosexual route, whereas K103N was seen in a 42-year-old monogamous female whose HIV sero-positive partner was initiated on ART 8 months back.
V106M is a nonpolymorphic mutation selected primarily by efavirenz (EFV) and causes high-level multi-NNRTI resistance especially in patients infected with HIV-1 subtype C. 9 In the HIV-1 mutation list for surveillance of NNRTI resistance developed by Shafer et al., there is no report of V106M among the ART-naive population as compared to 15% in treated individuals, indicating appearance of this mutation only on exposure to NNRTI. 10 Rajesh et al., in a prior study among HIV–TB coinfected patients, have also shown predilection of V106M toward the efavirenz-based regimen. 11 Thus, undisclosed exposure to NNRTI from private clinics cannot be ruled out, although V106M has been reported previously from India among ART-naive individuals. 8,12
The other NNRTI mutation, K103N, is also a nonpolymorphic mutation selected in patients receiving nevirapine (NVP) and EFV. It reduces NVP and EFV susceptibility by about 50- and 20-fold, respectively. 9,13 The lady denied nevirapine exposure during her previous pregnancies. In the absence of prior exposure, K103N may be a representative for persistence of TDR.
In India, prior studies on HIV drug resistance (HIVDR) among treatment-naive individuals have focused on TDR. Most studies from western and southern India have reported overall TDR prevalence of <5%. 14 –16 Conversely, a study in 2004 from western India has reported the presence of DRMs in 10% of sequences, which included resistance mutations against PIs. 17 However, recently, Azam et al. have reported zero prevalence of primary drug resistance-associated mutations to PIs in the north Indian city of Aligarh. 18
Earlier, Hamers et al., in a large sub-Saharan African cohort of people infected with HIV-1, indicated that the risk of virological failure and further acquisition of DRMs were more than double in patients with PDR. 19 Recently, Kantor et al. indicated that PDR and infection with subtype C were significantly associated with poor virological outcome. 20 HIV epidemic in India is driven by subtype C, which further highlights the role of periodic surveillance of PDR. The free ART program in India completed its decade of existence in 2014. The prevalence of PDR of 3.8% is well within the WHO threshold limit of <5% and indicates its successful implementation. To consolidate this achievement for a longer duration, it would be necessary to ensure optimal adherence to the regimen, counseling for safe sexual practices, efficient drug supply chain management, enhanced access to targeted plasma viral load test for prompt detection of treatment failure, and provision of second-line ART.
With a sample size of 52 and participants from a single center, this study deviated from the recently described WHO guidelines to assess HIVDR in adult populations to initiate ART. 21 Also use of the population-based sequencing method is known to miss the minority drug resistance variant. Nevertheless, this study provides a preliminary estimate of PDR after a decade of successful rollout of the free ART program in a part of western India.
Emergence and spread of DRMs among treatment-naive individuals can seriously compromise first-line regimen. In this study, we report an overall low prevalence of 3.8% of PDR restricted mainly to NNRTI. Absence of resistance against NRTI and PI reflects appropriateness of the currently recommended first-line regimen. This study supports the need for population-based surveillance of PDR.
Sequence Data
The GenBank Accession Nos. of 52 newly submitted partial pol gene sequences are from KT318883 to KT318934.
Footnotes
Acknowledgments
The authors would like to thank Dr. Sunil Zirpe, Dr. Mansa Angadi, Neelam Gurav, counselors, and nursing staff of Model Colony (NACO) ART center and all the study participants for their assistance. This work is supported by the intramural grant of National AIDS Research Institute (Indian Council of Medical Research), Pune.
Author Disclosure Statement
No competing financial interests exist.