Genetic Diversity in Pol Protein of HIV Type-1 Prevailing Among Injecting Drug Users in Pakistan

Abstract

Circulation of multiple subtypes (A, G and CRFs 02_AG) of human immunodeficiency virus type-1 (HIV-1) in select districts of Punjab province, Pakistan Owing to consistent genetic mutation and recombination, various escape mutants and/or drug-resistant mutants of human immunodeficiency virus (HIV-1) are emerging worldwide. Therefore, an understanding towards genomic characteristics of prevailing strains, particularly for drug resistance associated substitution, is very much essential to devise and implement necessary treatment and disease control intervention in disease endemic settings such as Pakistan. We processed a total of 130 plasma samples originating from HIV-treatment centers at select districts of Punjab province, Pakistan. The samples were first screened using HIV-1 Ag/Ab Combo test followed by amplification of pol gene (1084 bp) from samples positive either to antigen or both antigen and antibodies at once. Screening revealed a total of 45 samples positive (34.62%; 95% CI: 26.99-43.13) either to antigen or both antigen and antibodies at once (n = 18, 40%; 95% CI: 27.02-54.55) and antibodies alone (n = 27, 60%; 95% CI: 45.45-72.98). A maximum number of samples were positive from district Lahore [n= 19/43 (44.18%; 95% CI: 30.44-58.9)] followed by Faisalabad, n= 12/36 (33.33%; 95% CI: 20.21-49.66), Gujranwala, n= 05/23 (21.7%; 95% CI: 9.66-41.9) and Sargodha, n= 09/28 (32.1%; 95% CI: 17.93- 50.66). Odd of occurrence of HIV was significantly associated with individuals having a history of injecting drug use (68.08%; OR = 11.15; 95% CI: 53.84-79.61, p = 0.0001). The pol gene based phylogeny clustered study sequences into three distinct clades representing recombinant form 02_AG (n = 14, 77.0%; 95% CI: 54.79-91.00), subtype A (n = 2, 11.1%; 95% CI: 3.1-32.8) and G (n = 2, 11.1%; 95% CI: 3.1-32.8). Although we screened 18 samples for genotypic drug resistance analysis, except for an accessory mutation (M46K) in the protease (PR) region of a subject, we found a lack of drug resistance associated substitution in the PR. On the other hand, we found two subject (2/18) carrying resistance associated mutation (V106I) conferring a low level resistance against reverse transcriptase inhibitors. The present study concludes circulation of multiple subtypes of HIV-1 among affected population ascertaining a continuous disease surveillance coupled with evaluation of drug resistance at a higher resolution in future studies. Keywords: Human immunodeficiency virus type 1 (HIV-1); subtypes; co-circulation; molecular epidemiology; recombination analysis; drug resistance analysis Prevalence, molecular epidemiology and drug resistance pattern of human immunodeficiency virus type 1 among injecting drug users in Lahore, Pakistan Human immunodeficiency virus 1 (HIV-1) is common among injecting drug users (IDUs), and HIV- 1 positive IDUs may have potential of HIV transmission among general population through different ways. Therefore, an understanding towards current status of HIV-1 prevalence and genomic characteristics of circulating strains is crucial to devise and implement necessary interventions to control disease in developing countries like Pakistan. Total 201 plasma samples from IDUs were collected. Samples were first screened using HIV-1 Ag/Ab Combo test and then antigen positive samples were amplified for HIV-1 pol gene (1084 bp) and analyzed. Initial screening showed a total of 49 HIV samples positive (24.37%; 95% CI: 18.96-30.76). A substantial association of HIV incidence was observed in individuals with HCV infection (36.84%; 95% CI: 28.55-45.99; p<0.0001) followed by individuals involved in practices of shared injection equipment (21.17%; 95% CI: 25.07- 40.21; p<0.0001), injected previously used syringes (30.2%; 95% CI: 23.4-37.99; p=0.0016), sex with IDUs (37.78%; 95% CI: 25.11-52.37; p=0.002) and those with an age between 30-39 years (27.17%; 95% CI: 19.13-37.04; p=0.039). The pol gene-based phylogeny and subtyping classification categorized the under-study sequences representing subtype A (n = 12; 46.15%), CRF02_AG (n = 6; 23.08%), subtype C (n= 5; 19.23%) and subtype G (n = 3; 11.54%). Subsequent to genotyping resistance interpretation algorithm, one major (M46L) and two accessory (N88D, L89V) PIs mutations in the protease region while four NRTI (D67T, K70R/Q, M184V and T215F) and four NNRTI (V108T, E138A, V179I and Y181C) mutations in the reverse transcriptase region were observed. The present study concludes circulation of multiple subtypes of HIV-1 among IDUs and a continuous disease surveillance coupled with delineation of disease risk factors may provide a crucial insight into HIV prevention and treatment which could substantially curtail HIV epidemics in IDUs.