HIV-1 pol diversity among female bar and hotel workers in Northern Tanzania

Abstract

A national ART program was launched in Tanzania in October 2004. Due to the existence of multiple HIV-1 subtypes and recombinant viruses co-circulating in Tanzania, it is important to monitor rates of drug resistance. The present study determined the prevalence of HIV-1 drug resistance mutations among ART-naive female bar and hotel workers, a high-risk population for HIV-1 infection in Moshi, Tanzania. A partial HIV-1 pol gene was analyzed by single-genome amplification and sequencing in 45 subjects (622 pol sequences total; median number of sequences per subject, 13; IQR 5-20) in samples collected in 2005. The prevalence of HIV-1 subtypes A1, C, and D, and inter-subtype recombinant viruses, was 36%, 29%, 9% and 27%, respectively. Thirteen different recombination patterns included D/A1/D, C/A1, A1/C/A1, A1/U/A1, C/U/A1, C/A1, U/D/U, D/A1/D, A1/C, A1/C, A2/C/A2, CRF10_CD/C/CRF10_CD and CRF35_AD/A1/CRF35_AD. CRF35_AD was identified in Tanzania for the first time. All recombinant viruses in this study were unique, suggesting ongoing recombination processes among circulating HIV-1 variants. The prevalence of multiple infections in this population was 16% (n = 7). Primary HIV-1 drug resistance mutations to RT inhibitors were identified in three (7%) subjects (K65R plus Y181C; N60D; and V106M). In some subjects, polymorphisms were observed at the RT positions 41, 69, 75, 98, 101, 179, 190, and 215. Secondary mutations associated with NNRTIs were observed at the RT positions 90 (7%) and 138 (6%). In the protease gene, three subjects (7%) had M46I/L mutations. All subjects in this study had HIV-1 subtype-specific natural polymorphisms at positions 36, 69, 89 and 93 that are associated with drug resistance in HIV-1 subtype B. These results suggested that HIV-1 drug resistance mutations and natural polymorphisms existed in this population before the initiation of the national ART program. With increasing use of ARV, these results highlight the importance of drug resistance monitoring in Tanzania.

Figure 1. ML phylogenetic tree of HIV-1 protease and reverse transcriptase sequences of the pol gene from 45 subjects.

The ML was constructed by PhyML 3.0.1 and visualized in FigTree. The tree is rooted with the HIV-1 group N consensus sequence as an outgroup. 488 non-recombinant pol sequences generated from the 45 subjects were analyzed with HIV-1 reference subtypes from the Los Alamos HIV-1 Sequence Database. HIV-1 inter-subtype recombinant pol DNA sequences were excluded in this figure. HIV-1 reference subtypes A1, C, and D are shown in red, pink and blue, respectively. The other references are shown in orange. Approximate likelihood ratio test (aLRT) values of ≥0.95 were considered significant and are shown by an asterisk (*). The scale at the bottom of the figure corresponds to 0.01 nucleotide substitutions per site.

Figure 2. Schematic representation of recombinant viruses detected in 12 subjects showing recombination breakpoints.

Localization of breakpoints between HIV-1 subtypes was done by SimPlot analysis and identified breakpoints were visually inspected in BioEdit. The numbers in each bar indicate breakpoints and were given HXB2 numbering. The first bar represents the protease and reverse transcriptase genes. The dashed lines indicate the positions where protease gene starts, 2, 253 (HXB2 numbering), and ends, 2,549 (HXB2 numbering). The genome segments are colored according to HIV-1 subtypes. Red, orange, yellow, blue, green, and light blue represent sequences from HIV-1 subtypes A1, A2, C, D, CRF10_CD and CRF35_AD, respectively. The black bar represents the unclassified (U) regions. Confirmation of recombination was conducted by constructing phylogenetic trees of the putative recombinant regions against reference HIV-1 subtypes.

Figure 3. Polymorphisms in protease gene in HIV-1 subtypes A1 and C, compared to ARV-naïve individuals from the Stanford HIV-1 Drug Resistance Database.