Phylogenetic Analysis of Human Immunodeficiency Virus from People Who Inject Drugs in Indonesia, Ukraine, and Vietnam: HPTN 074

Abstract

Background: HIV Prevention Trials Network (HPTN) 074 evaluated human immunodeficiency virus (HIV) prevention interventions for people who inject drugs (PWID) in Indonesia, Ukraine, and Vietnam. Study interventions included support for HIV infection and substance use treatment. The study enrolled index participants living with HIV and injection partners who were not living with HIV. Seven partners acquired HIV infection during the study (seroconverters). We analyzed the phylogenetic relatedness between HIV strains in the cohort and the multiplicity of infection in seroconverters.

Methods: Pol region consensus sequences were used for phylogenetic analysis. Data from next-generation sequencing (NGS, env region) were used to evaluate genetic linkage of HIV from the 7 seroconverters and the corresponding index participants (index-partner pairs), to analyze HIV from index participants in pol sequence clusters, and to analyze multiplicity of HIV infection.

Results: Phylogenetic analysis of pol sequences from 445 index participants and 7 seroconverters identified 18 sequence clusters (2 index-partner pairs, 1 partner-partner pair, and 15 index-only groups with 2-7 indexes/cluster). Analysis of NGS data confirmed linkage for the 2 index-partner pairs, the partner-partner pair, and 11 of the 15 index-index clusters. The remaining 5 seroconverters had infections that were not linked to the corresponding enrolled index participant. Three (42.9%) of the 7 seroconverters were infected with more than 1 HIV strain (3-8 strains per person).

Conclusions: We identified complex patterns of HIV clustering and linkage among PWID in 3 communities. This should be considered when designing strategies for HIV prevention for PWID.

Clinical trials registration: NCT02935296.

Keywords: HIV; multiplicity of infection; people who inject drugs; phylogenetic analysis.

© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Figures

Figure 1.

The HIV Prevention Trials Network 074 study cohort. The flow chart provides an overview of the study cohort, indicating the number and source of HIV sequences used in the analysis. One index participant did not have a sample from the enrollment visit available for testing (indicated with an asterisk); in that case, a sample collected at the screening visit was used for analysis. Abbreviations: HIV, human immunodeficiency virus; VL, viral load.

Figure 2.

Phylogenetic trees of study and background pol sequences from each study site. Phylogenetic trees were generated using pol gene sequences generated by population sequencing. Trees were generated using RAxML for each study site: (A) Indonesia, 109 study sequences; (B) Ukraine, 169 study sequences; and (C) Vietnam, 188 study sequences. Gray dots corresponding to tree branches represent background (control) sequences obtained from a sequence database (see the Methods section). Dark blue dots represent study sequences from index participants. Light blue dots represent study sequences from injection partners who acquired human immunodeficiency virus (HIV) infection during the study (seroconverters). The letter “C” indicates a cluster of 2 or more index participants (1 sequence per person). The letter “I” indicates sequences from the 7 index participants whose partners acquired HIV infection during the study (I1–I7; 2 sequences per person). The letter “P” indicates sequences from the 7 seroconverters (P1–P7; 2 sequence per person). Numbers indicate that the index and partner were enrolled as part of an injection group (eg, index I1 was enrolled with partner P1). Brackets indicate paired sequences from the same individual obtained from samples collected at different study visits. Branches for sequences in clusters are highlighted with shading. Purple shading indicates index sequences that are part of an index-partner cluster (participants I1–I7); light blue shading indicates seroconverter sequences (participants P1–P7); gray shading indicates clusters of sequences from 2 or more index participants (clusters C1–C15). Note that the cluster designated C15 includes 7 index participants and 2 nonstudy background sequences (C). Abbreviations: I, index; P, partner; C, cluster; RAxML, Randomized Axelerated Maximum Likelihood.

Figure 3.

Phylogenetic trees of env sequences from index-partner (IP) pairs and a partner-partner (PP) pair with genetically linked human immunodeficiency virus (HIV) infections. Phylogenetic trees were generated using env sequences from next-generation sequencing for 7 seroconversion cases (IP pairs, A–G) and 1 PP cluster (H) are shown. Colors indicate the source of study sequences, as noted in the lower right of the figure. Each tree includes nonstudy HIV env sequences from each participating country and background sequences (black branches). Bootstrap values were obtained for 1000 replicate trees; values ≥90% are shown. Case numbers (IP1–IP7, PP) refer to cases described in Table 1. In 1 case (IP1), the partner sequences clustered with low-level viral variants present in 1 index sample; these variants represented approximately 5% of the env read counts in the index sample.

Figure 4.

Phylogenetic trees of env sequences from a subset of the index-index clusters. Phylogenetic trees generated using env gene sequences from next-generation sequencing are shown for 4 index-index clusters (see Table 1): a representative unlinked case (cluster C14; A), a representative linked case (cluster C7; B), the cluster involving 3 index participants (cluster C12; C), and the cluster involving 7 index participants (cluster C15; D). Colors indicate the source of study sequences, as noted in the upper left of each panel. Each tree includes nonstudy human immunodeficiency virus env sequences from each participating country and background sequences (black branches). Bootstrap values were obtained for 1000 replicate trees; values ≥90% are shown. Case numbers (C14, C7, C12, and C15) refer to cases described in Table 1.

Figure 5.

Analysis of within-individual env diversity. The figure shows representative neighbor-joining phylogenetic trees (top panels), Highlighter plots (middle panels), and histograms of the distribution of pairwise genetic distances (lower panels). Data are shown for a representative participant infected with a single human immunodeficiency virus (HIV) variant (P5; A) and a representative participant infected with multiple HIV variants (P6; B). Highlighter plots show the number of sequence mismatches as a function of nucleotide position (env alignment position) compared with an intrahost consensus sequence (master). Nucleotide mismatches are shown using the following colors: thymine, red; adenine, green; cytosine, blue; and guanine, orange. The histograms show the distribution of within-individual pairwise genetic distance; the red vertical line shows the median value. The same results were obtained when the analysis was performed using maximum-likelihood (RAxML) phylogenetic trees. Abbreviation: m, master.