Basic demographic and baseline clinical characteristics of 103 HIV-1 patients initiating cART in Kuala Lumpur, Malaysia.

<p>Categorical variables are described as n (%) and numerical variables are described as mean (± SD) or median (IQR).</p><p>Abbreviations: HIV-1, human immunodeficiency type-1; cART, combination antiretroviral therapy; SD, standard deviation; IQR, interquartile range.</p><p>^ψ<i>P</i> values are given for test of general differences across the two subgroups using an independent t-test^€ for normally-distributed categorical outcomes and Mann-Whitney U test^Δ for non-normally-distributed continuous outcomes (<i>P</i> values of < .05 are considered significant).</p><p>^a data includes Malay, Indian and other minor ethnicities.</p><p>^b data includes men-who-have-sex-with-men (MSM), unsafe injecting drug use and blood transfusion.</p><p>^c the interval between the day of cART initiation and the last clinical consultation available.</p><p>^d includes co-infections with tuberculosis, hepatitis B or C virus (HBV/HCV).</p><p>Basic demographic and baseline clinical characteristics of 103 HIV-1 patients initiating cART in Kuala Lumpur, Malaysia.</p

Kaplan-Meier analysis of time to reach CD4+ T-cell count of ≥350 cells/μL (<i>A</i>) or to ≥500 cells/μL (<i>B</i>) in patients infected with CRF01_AE and subtype B on cART.

<p><i>P</i> values were calculated using log-rank test.</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>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

Bootscan and informative sites analyses of the 1.6kb partial <i>gag-pol</i> gene sequences of various HIV-1 unique recombinant forms (URFs) characterised among the blood donors in Kuala Lumpur.

A, Bootscan plot of strain 14MYNBB230 which shares all four recombination breakpoints with a previously reported URF, 05MYKL043 (GenBank accession number: DQ366666) [14]. Both strains also share two identical breakpoints with CRF33_01B, as denoted by square textboxes. B, Bootscan plot of strain 13MYNBB034 which displays a total of six recombinant segments involving CRF01_AE and subtype B' (of Thai origin), whereby three breakpoints were identical to that of CRF33_01B and CRF74_01B (which shares four breakpoints with CRF33_01B). Strain 13MYNBB034 may be another newly-characterised genetic variant of CRF33_01B which differed structurally from CRF74_01B. C, Bootscan plots of four CRF01_AE/B' URFs (13MYNBB048, 13MYNBB128, 13MYNBB059 and 14MYNBB193) displaying distinct mosaic recombination structures and breakpoints, which have yet to be reported in the country. D, Bootscan plots of subtype B'/C recombinants displaying all identical recombination structures and breakpoints amongst each other which potentially represent a novel CRF candidate in Kuala Lumpur. All four strains which are epidemiologically-unlinked (13MYNBB108, 14MYNBB084, 14MYNBB090 and 14MYNBB164) also shared three breakpoints (as denoted by square textboxes) with CRF07_BC which is prevalent in China [32]. These isolates, however were genetically and structurally distinct from CRF08_BC from China. E, Bootscan plots of subtype B'/G recombinants (13MYNBB064 and 13MYNBB065) displaying all identical recombination structures and breakpoints amongst each other. Putative HIV-1 parental reference genotypes were selected by similarity plot, which included 90THCM235 (CRF01_AE), CNRL42 (subtype B' of Thai origin), 95IN21068 (subtype C) and 01NGPL0674 (subtype G). All breakpoints were labelled numerically and identical breakpoints were highlighted with dotted lines. Bootscan was performed in SimPlot version 3.5.1 [24] using a window size of 200 nucleotides moving along the alignment in increments of 20 nucleotides.</p

Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries - Fig 3

<p><b>Sub-region neighbour joining tree analyses of the 1.6kb partial <i>gag-pol</i> genes sequenced in two clusters of (A) subtype B'/C and (B) B'/G recombinants characterised in the population.</b> Based on the informative sites analyses, recombination breakpoints were estimated for each strain and the partial <i>gag-pol</i> sequences (HXB2:1753–3440) were then sub-divided into different regions for phylogenetic reconstruction. Putative HIV-1 parental reference genotypes used in bootscan were 90THCM235 (CRF01_AE), CNRL42 (subtype B' of Thai origin), 95IN21068 (subtype C) and 01NGPL0674 (subtype G). Incident or prevalent HIV-1 infections for each strain, as determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) were identified by orange triangles or red circles, respectively. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 1% genetic distance (0.01 substitutions per site).</p

Genetic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF74_01B) Identified among Intravenous Drug Users in Malaysia: Recombination History and Phylogenetic Linkage with Previously Defined Recombinant Lineages

<div><p>In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs). Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs), especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the <i>gag-pol</i> and <i>nef</i> regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype Bʹ that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region.</p></div

Phylogenetic reconstruction of 136 partial <i>gag-pol</i> gene sequences of 1.6kb amplified among the blood donors in Kuala Lumpur, Malaysia between 2013 and 2014.

<p>HIV-1 incidence was estimated using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) to identify recent (incident) and long-standing (prevalent) infections as indicated where available. Neighbour-joining tree was constructed in MEGA 5.05 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161853#pone.0161853.ref022" target="_blank">22</a>] using Kimura 2-parameter method of nucleotide substitutions and the reliability of the branching nodes were assessed by bootstrap analysis of 1000 replicates. Eleven partial <i>gag</i>-PR (834bp) and two RT gene sequences (966bp) were genotyped separately using similar methods and their prevalence was reported in this study (figures not shown for clarity). Relevant HIV-1 reference genotypes in Southeast Asia include subtype B, CRF01_AE, CRF33_01B, CRF34_01B, CRF48_01B, CRF52_01B, CRF53_01B, CRF54_01B, CRF58_01B and CRF74_01B. Reference sequences of other genotypes prevalent in China (CRF07_BC, CRF08_BC and other recently-described B'/C CRFs) and Africa (subtype G, CRF02_AG and CRF45_cpx) were also included in the analysis. The reference sequences were labelled in the following order: genotype, country of origin, isolate name and GenBank accession number. A well-supported cluster of Malaysian subtype G strains was also highlighted as G_MY within the subtype G clade of African reference strains. All 12 unique recombinant forms were denoted by closed diamonds and labelled according to incident or prevalent infection status. Clusters of novel B'/C recombinants (strains 13MYNBB108, 14MYNBB084, 14MYNBB090 and 14MYNBB164) and B'/G recombinants (13MYNBB064 and 13MYNBB065) were highlighted in the tree. Simian immunodeficiency virus (SIVcpz) reference strains were included as outgroup. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 1% genetic distance (0.01 substitutions per site).</p

HIV-1 genotype distribution of incident and prevalent infections among 127 blood donors in Kuala Lumpur.

<p>A limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) was used to distinguish incident from prevalent HIV-1 infections. Out of 179 samples available for incidence assay testing, 70.9% (n = 127) were successfully genotyped and comprised of 29 (22.8%) incident HIV-1 infections.</p

Demographic characteristics of study subjects infected by HIV-1 CRF74_01B.

<p>* IDU, intravenous drug users</p><p>^# Partial genome</p><p>Demographic characteristics of study subjects infected by HIV-1 CRF74_01B.</p

Recombination structures and sub-genomic maximum clade credibility tree reconstructions.

<p>Recombination structures of HIV-1 CRF33_01B, CRF53_01B and CRF74_01B were shown. Shared recombination breakpoints between these CRFs were indicated by dashed lines. Maximum clade credibility trees for the <i>gag</i> region (region I; HXB2: 790–2052) and <i>pol-env</i> region (region VII; HXB2: 3326–9012) of CRF74_01B and other relevant reference sequences were reconstructed. The means of tMRCA of CRF74_01B for region I and region VII were estimate around 1996.2 and 1993.9, respectively, with the 95% credibility intervals stated in parentheses.</p