Cox proportional hazards using unadjusted (univariate) and adjusted (multivariate) analyses to assess the effects of covariates on the rate of CD4+ T cell recovery, defined as time for CD4+ T-cell count increase to ≥350 cells/μL during cART.

<p>Abbreviations: cART, combination antiretroviral therapy; HR, hazard ratio; CI, confidence intervals; Ref, reference.</p><p>^a covariates with P values < .25 were included in the adjusted analysis. The rate of CD4+ T cell recovery was also adjusted for baseline HIV-1 RNA because it is known to be an important predictor of immunologic recovery.</p><p>Cox proportional hazards using unadjusted (univariate) and adjusted (multivariate) analyses to assess the effects of covariates on the rate of CD4+ T cell recovery, defined as time for CD4+ T-cell count increase to ≥350 cells/μL during cART.</p

Molecular Diversity of HIV-1 among People Who Inject Drugs in Kuala Lumpur, Malaysia: Massive Expansion of Circulating Recombinant Form (CRF) 33_01B and Emergence of Multiple Unique Recombinant Clusters

<div><p>Since the discovery of HIV-1 circulating recombinant form (CRF) 33_01B in Malaysia in the early 2000 s, continuous genetic diversification and active recombination involving CRF33_01B and other circulating genotypes in the region including CRF01_AE and subtype B′ of Thai origin, have led to the emergence of novel CRFs and unique recombinant forms. The history and magnitude of CRF33_01B transmission among various risk groups including people who inject drugs (PWID) however have not been investigated despite the high epidemiological impact of CRF33_01B in the region. We update the most recent molecular epidemiology of HIV-1 among PWIDs recruited in Malaysia between 2010 and 2011 by population sequencing and phylogenetic analysis of 128 <i>gag-pol</i> sequences. HIV-1 CRF33_01B was circulating among 71% of PWIDs whilst a lower prevalence of other previously dominant HIV-1 genotypes [subtype B′ (11%) and CRF01_AE (5%)] and CRF01_AE/B′ unique recombinants (13%) were detected, indicating a significant shift in genotype replacement in this population. Three clusters of CRF01_AE/B′ recombinants displaying divergent yet phylogenetically-related mosaic genomes to CRF33_01B were identified and characterized, suggestive of an abrupt emergence of multiple novel CRF clades. Using rigorous maximum likelihood approach and the Bayesian Markov chain Monte Carlo (MCMC) sampling of CRF33_01B_pol sequences to elucidate the past population dynamics, we found that the founder lineages of CRF33_01B were likely to have first emerged among PWIDs in the early 1990 s before spreading exponentially to various high and low-risk populations (including children who acquired infections from their mothers) and later on became endemic around the early 2000 s. Taken together, our findings provide notable genetic evidence indicating the widespread expansion of CRF33_01B among PWIDs and into the general population. The emergence of numerous previously unknown recombinant clades highlights the escalating genetic complexity of HIV-1 in the Southeast Asian region.</p></div

Sub-region trees analyses of the unique recombinant clusters.

<p>The <i>gag-pol</i> genes were sequenced, codon-aligned and manually adjusted with the HIV-1 reference subtypes and circulating recombinant forms (CRFs) retrieved from the Los Alamos HIV database (<a href="http://www.hiv.lanl.gov/" target="_blank">http://www.hiv.lanl.gov/</a>). The 1.6kb <i>gag-pol</i> sequences of the unique recombinant clusters were subdivided into regions (denoted by Roman letters) in relation to the unique recombination breakpoints estimated by informative site analysis. Sub-region neighbour-joining trees were constructed in MEGA 5.05 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone.0062560-Tamura1" target="_blank">[34]</a> using Kimura 2-parameter method for nucleotide substitutions to estimate pair-wise evolutionary distance. The reliability of the branch nodes were assessed by bootstrap analysis of 1000 replicates. Bootscan plots and sub-region neighbour-joining trees for each CRF candidates were shown: <b>A</b>, novel CRF candidate 1 (10MYKJ036, 11MY1RJ704, 11MY1ZK731 and 11MY1EP794), <b>B</b>, novel CRF candidate 2 (10MYKJ067, 10MYKJ079 and 04MYKL016) and <b>C</b>, novel CRF candidate 3 (10MYKJ016, 10MYKJ052, 11MYIYC672 and 11MY1JJ741). HIV-1 reference strains CRF01_CM240 (CRF01_AE) and B′_RL42 (Thai subtype B′) were selected as the putative parental genotypes by similarity plotting and included in the bootscan plots (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone-0062560-g002" target="_blank"><b>Figure 2</b></a>). The novel CRF candidate 1 or CRF33_01B were highlighted where the sequences formed a clade in the respective sub-region trees. In cases where the sub-region of CRF33_01B clustered with the reference strains of either putative parental genotypes - CRF01_AE or B′ showing the parental origin, the parental genotype was highlighted. The HXB2 nucleotide positions and sequence length of individual sub-regions were indicated in the illustration. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar for respective sub-region trees was also indicated (in substitutions per site).</p

Bootscan analyses of HIV-1 unique recombinant forms (URFs) isolated from people who inject drugs (PWID) in Malaysia.

<p><b>A</b>, Schematic representation of the full length recombinant structure of CRF33_01B with the four unique recombination breakpoints labelled as 1–4 (HXB2∶2053–2063 nt, 2375–2416 nt, 2538–2540 nt and 2841–2875 nt, respectively) in the <i>gag-pol</i> region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone.0062560-Tee3" target="_blank">[21]</a>. <b>B</b>, Bootscanning plots of three novel CRF candidates (1 to 3) generated from the 1.6 kb <i>gag-pol</i> gene (HXB2∶1753–3440 nt) using SimPlot version 3.5.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone.0062560-Lole1" target="_blank">[36]</a>. HIV-1 reference strains CRF01_CM240 (CRF01_AE) and B′_RL42 (Thai subtype B′) were selected as the putative parental genotypes by similarity plotting and C_95IN21068 (subtype C) as an outgroup, with a window size of 200 nucleotides moving along the alignment in increments of 50 nucleotides to define the recombination structures. The shared recombination breakpoints between the URFs were highlighted and in cases where the breakpoints were also shared with CRF33_01B, these breakpoints were further numbered (1 to 4). Each sub-region of these CRF candidates was also indicated and described further in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone-0062560-g003" target="_blank"><b>Figure 3</b></a>. <b>C,</b> Bootscanning plots of unique recombinant forms (URFs) –11MY1MR851 (sharing a similar recombination breakpoint with 07MYKLD49 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone.0062560-Lau2" target="_blank">[46]</a>), 10MYKJ086, 11MY2NF831, 11MY3HA741 and 11MY1IM781 displaying distinct recombinant structures in the 1.6 kb <i>gag-pol</i> sequences amplified among the PWIDs study population. For clarity, bootscanning plots of partial RT and <i>gag-</i>PR sequences (11MY1JJ741 and 10MYKJ084, respectively) were not displayed here but their recombination structures have been discussed in detail in the text.</p

Phylogenetic analysis of the 1.6 kb HIV-1 <i>gag-pol</i> sequences (HXB2∶1753–3440) amplified among people who inject drugs (PWID) in Kuala Lumpur, Malaysia.

<p>The <i>gag-pol</i> genes (n = 98) were sequenced, codon-aligned and manually adjusted with the HIV-1 reference subtypes and circulating recombinant forms (CRFs) retrieved from the Los Alamos HIV database (<a href="http://www.hiv.lanl.gov/" target="_blank">http://www.hiv.lanl.gov/</a>). Neighbour-joining tree was constructed in MEGA 5.05 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062560#pone.0062560-Tamura1" target="_blank">[34]</a> using Kimura 2-parameter method of nucleotide substitutions to estimate pair-wise evolutionary distance and the reliability of the branching nodes were assessed by bootstrap analysis of 1000 replicates. Reference strains of established and informative HIV-1 genotypes – CRF01_AE, subtype B (including Thai B′), CRF15_01B, CRF34_01B CRF33_01B, CRF48_01B, CRF51_01B, CRF52_01B and other relevant unique recombinant forms (URFs) circulating in Southeast Asia were also included in the analysis to improve tree resolution. Other HIV-1 genotypes – subtypes C, D and SIVcpz reference strains (CPZ.US_MARILYN and CPZ.SIVcpzCAM13) were included as outgroup. The newly emerged unique recombinant clusters identified in this study were highlighted as CRF candidate 1 (10MYKJ036, 11MY1RJ704, 11MY1ZK731 and 11MY1EP794), CRF candidate 2 (10MYKJ067, 10MYKJ079 and 04MYKL016) and CRF candidate 3 (10MYKJ016, 10MYKJ052, 11MY1YC672 and 11MY1JJ741). Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 2% genetic distance (0.02 substitutions per site).</p